ENERGY METABOLISM OF SCHISTOSOMES
by
Clement Antonio Earle B.Sc (Hons).
A thesis submitted for
the degree of Doctor of Philosophy
in'the University of London
and for the Diploma of Imperial College
Department of Pure and Applied Biology,
Imperial College of Science and Technology,London Slii7 2BB September, 1983
ABSTRACT
Studies on carbohydrate catabolism in several species of
adult schistosomes, in particular Schistosoma mansoni and
S.margrebowiei, have been carried out. The basic body constituents i.e. protein, glycogen and lipid, together with wet weight, were measured in these two species. The specific
activities of certain key glycolytic, tricarboxylic (TCA) cycle
and hexosemonophosphate (HMP) shunt enzymes have been measured in S.bovis, S.leiperi, S,haematobium and S,.japonicum to provide an indication of the relative significance of these pathways in
these parasites. A more detailed examination of these pathways
using enzyme activity measurements and determination of steady
state levels of intermediary metabolites has been performed for
5.mansoni and S.marqrebowiei.
The release of end products and changes in endogenous levels
of carbohydrate were measured during incubation experiments involving 5.mansoni, S.marqrebowiei, S.haematobium and
Schistosomatium douthitti to establish which catabolic pathways the parasites utilise. Additional studies included an assessment
of the regulatory role of pyruvate kinase in 5.mansoni, .
S.marqrebowiei, S.japonicum and S.bovis. A study of the nin vivo”
activity of the enzyme from 5.mansoni has been performed using
"physiological”Llevels of reactants, determined from the previously measured metabolite levels. Oxygen uptake measurements and
determination of cytochrome spectra have been carried out in
S,mansoni to establish the role of oxidative phosphorylation inthis species
nc.
The results indicate that in general, schistosomes rely
heavily on glycolysis for energy production and that the TCA and
HMP pathways appear to contribute little in terms of ATP synthesis.
The incubation experiments showed that female schistosomes channel
approximately half of their glucose consumption into lactate produce
tion. The fate of the remaining glucose is unknown but a signifi- ) cant proportion could be accounted for in S.mansoni by oxidative
phosphorylation coupled via a glycerol shuttle mechanism.
Enzyme kinetic studies indicate that pyruvate kinase is
playing a regulatory role in glycolysis in S.mansoni. The male
and female enzymes showed a differential response to fructose-1,
6- bisphosphate (FBP) but were inhibited to the same degree by ATP. Evidence for pyruvate kinase regulation in other schistosome
species is not conclusive.In general, schistosomes seem to have wider metabolic
capabilities for ATP production than previously assumed. The
female parasites in particular are not homolactic fermenters as
they channel half their glucose consumption into non-glycolytic
energy synthesis and possibly anabolic metabolism.
ACKNOWLEDGEMENTS
I would like to express my thanks to Dr. D.P. McManus for
his help and discussion throughout this study. I would also
like to thank Mr. R.J. Knowles of the British Museum of Natural
History for his kind provision of some of the schistosome material.
In addition, I am grateful to Dr. D. Hayes of St Bartholomew^
Biochemistry Department and Miss Sheila Lanham of the London
School of Hygiene and Tropical Medicine for their assistance andoadvice regarding the cytochrome and electrophretic analyses.
I am indebted to Mr. D. Featherston, Mr. R.J. Cripps and
Mrs. J Crombie for their constant friendship and enthusiastic support. Also, I would like to thank the Science and Engineering
Research Council, Miss S.E. Revington and my mum and dad for their
financial assistance and encouragement throughout this project. Finally, I am extremely grateful to Mrs. J. True for her patience,
skill and generosity in typing this manuscript.
CONTENTS
CHAPTER PAGE NO.
Abstract 1
Acknowledgements 3
1. Introduction 1 o
1.1 General Introduction 10
1.2 Energy metabolism in adult schistosomes 12
1.2.1. Basic considerations 12
1.2.2. Aims 18
2. Materials and methods 20
2.1. Maintenance 20
2.1.1. Snails 20
2.1.2. Mice 202.1.3 Infections 202.1.3.1. Egg collection, hatching and collection
of miracidia 20
2.1.3.2. Snail infections 21
2.1.3.3. Collection of cercariae 21
2.1.3.4. Mouse infections 222.1.4. Recovery of adult parasites 23
2.2. Chemicals 24
2.3. Carbohydrate and wet weight determinations 242.4. Protein determinations 252.5. Lipids 26
PAGE NO
2.6* Enzyme assays 272.7. Metabolites 382.7.1. Spectrophotometry 382.7.1.1. Preparation of material 382.7.1.2. Assays 392.7.2. Fluorimetry 402.7.2.1. Preparation of material 402.7.2.2. Assays 412.8. Incubations 432.8.1. End product analysis 452.8.1.1. Medium 452.8.1.2. Endogenous metabolite levels 492.8.1.3. ^ C 02 measurement 502.8.2. Glucose incorporation 512.9. Pyruvate kinase kinetics 522.9.1. Physiological enzyme activity 532.9.1.1. Male enzyme 532.9.1.2. Female enzyme 532.9.2. Effects of M(f+/Mrt'+ on enzyme activity
and enzyme stability 542.9.3. Host enzyme activity in parasite gut
contents 542.9.4. Mouse erythrocyte and serum pyruvate
kinase 55
PAGE NO
*
*
*
*
♦
*
2.10. Electrophoresis 56
2.10.1. PAGE 562.10.2. Starch gel 57
2.10.3. Cellulose-acetate 57
2.10. A. Isoelectric focusing 56
2.11. Cytochromes 61
2.12. Oxygen uptake 63
3. Results 64
3.1. Maintenance and collection of parasites 64
3.1.1. Molluscan hosts 643.1.2. Infections 64
3.1.2.1. Recovery of adult parasites 653.2. Statistical analyses 653.3. Carbohydrate determination 57
3.3.1. Glucose levels 673.3.2. Glycogen levels 68
3.4. liJet weight determinations 71
3.5. Protein determinations 71
3.6. Protein : Wet weight ratios 72
3.7. Total lipids 78
3.8. Enzyme analysis 783.8.1. S.mansoni and S.marqrebowiei 783.8.1.1. Glycolysis 78
PAGE NO
3.8.1.2. TCA cycle 79
3.8.1.3. HMP shunt 79
3.8.2. Enzyme activities in other species 79
3.9. Metabolites 87
3.9.1. Analysis of metabolites of mouse liver 87
3.9.2. Analysis of standard metabolite solutions 873.9.3. Analysis of parasite-..metabolite levels 90
3.9.3.1. S.mansoni 90
3.9.3.2. S.marqrebouiei 903.9.4. Mass action ratios 94
3.10. Incubations 94
3.10.1. S.mansoni 943.10.1.1. Glucose uptake and lactate production 94
3.10.1.2. Metabolite levels 95143.10.1.3. CO2 production 95
3.10.1.4. Glucose incorporation 99
3.10.2. S.marqrebouiiei 99
3.10.2.1. Glucose uptake and lactate production 99
3.10.2.2. Metabolite levels 102143.10.2.3. CO2 production 102
3.10.3. 5.haematobium 102
3.10.3.1. Glucose uptake and lactate production 1023.10.3.2. Metabolite levels 103
3.10.4. S.douthitti 103
103
3.10.4.2. Metabolite levels 103
3.10.4.3. ^ C 02 production 1063.10.5. Interspecific comparisons 106
3.11. Kinetic study of pyruvate kinase 1073.11.1. S.mansoni 107
3.11.1.1. ATP inhibition 111
3.11.1.2. FBP activation 111
3.11.2. S.marqrebouiiei 112
3.11.2.1. ATP inhibition 112
3.11.2.2. FBP activation 1123.11.3. S..japonicum 115
3.11.4. S.bovis 115
3.11.5. Interspecific comparisons 123
3.11.6. Pyruvate kinase activity under physiological
substrate and modulator concentrations 1233.11.6.1. Enzyme activity in parasite gut contents 1243.11.6.2. Kinetics of PK from male S.mansoni 124
3.11.6.3. Kinetics of PK from female S.mansoni 126
3.11.6.4. Stability and effect of cations on theactivity of PK 150
3.11.6.5. Pyruvate kinase from mouse erythrocyte 1533.12. Electrophoresis 1533.12.1. Isoelectric focusing 157
3 ,1 0 .4 .1 G lucose uptake and la c ta te prod uction
PAGE NO.
3.13. Cytochromes 1373.13.1. Controls 1373.13.1.1. Whole mouse blood and rat liver 1373.13.1.2. Digested mouse blood 1383.13.1.3. Haematin 1383.13.2. S.mansoni 1383.13.2.1. Males 1423.13.2.2. Females 1423.14 Oxygen uptake 1424. DISCUSSION 1484.1. Glucose, glycogen, protein, uiet weight and
lipid levels 1484.2 Enzyme analysis 1514.3. Metabolites 1544.4. Incubations 1564.5. Pyruvate kinase 1654.6. Oxygen uptake and cytochromes 1734.7. Summary 180
References 183»
CHAPTER 1
INTRODUCTION
1.1. GENERAL INTRODUCTION
Schistosomiasis is one of the most prevalent parasitic
diseases in man. Currently, it is second only to malaria in
terms of public health importance in tropical and sub-tropical
regions. The disease is now endemic in seventy-three countries
and recent estimates indicate that nearly 500 million people world wide are at risk of infection (Iarotski & Davis, 1981). In
contrast to malaria, little advance has been made with regard
to the chemotherapy of schistosomiasis since the introduction of antimonials in 1918 (Huang, 1981). Further studies of
schistosome biochemistry are suitable, as comparatively little
detail is known concerning the basic metabolism of these parasites
Moreover, conflicts of opinion exist regarding certain aspects of
the available data. A clearer knowledge of basic schistosome
metabolism may eventually result in wider, more rational methods of drug control.
At present, Schistosoma mansoni is the most widely studied
species, primarily because of its pathogenicity and the relative
ease with which it can be maintained in the laboratory. The
general schistosome life cycle is shown in Fig.T. There are two parasitic and free living stages in the cycle and it has been
*
Figure 1.A
The general life cycle cf schistosomes.*
%
*
<*
ft
Adult host
Adult parasitesEggs retained in tissues
A
Common environment in water
Free swimming Cercariae
Free swimming Miracidium
Intermediate host
demonstrated that the energy metabolism of the different stages
alters in relation to the helminth's environment.
The miracidial and cercarial stages are capable of operating
a fully oxidative metabolism (Bruce et al, 1971; Coles, 1972 b,
1973a). As these forms are free living, oxidative metabolic
processes are a more efficient means of coupling the greater
oxygen content of the external environment to energy synthesis.
Examination of sporocyst (intramolluscan stage) metabolism is
complicated due to the difficulties of separating the parasite from host tissue. Consequently details of sporocyst metabolism
are lacking.On invasion of the definitive (adult) host the cercaria moves
into an environment with a lower oxygen tension. After penetration,
the cercaria becomes a schistosomulum and there is a change from
aerobic metabolism to anaerobic lactate production (Coles, 1972a, 1973b, 1973c; Bruce et al, 1969; Lawson & Wilson, 1980). The
schistosomulum migrates via the tissues and blood system to the
final adult sites which are usually the portal or mesenteric
vessels.
1.2. ENERGY METABOLISM IN ADULT SCHISTOSOMES
1.2.1• Basic considerationsAdult helminths are totally reliant on carbohydrate as a
source of metabolic fuel. They are also generally unable to
catabolise glucose completely to carbon dioxide (C0£) and water, hence they produce a variety of reduced organic end-products
*
♦
♦
♦
*
♦
*
(Barrett, 1981). Schistosomes take up glucose (Fripp, 1967;Uglem & Read, 1975; Cornford & Oldendorf, 1979) and also contain
large quantities of glycogen (Bueding & Koletsky, 1950; Lennox
& Schiller, 1972; Magzoub, 1974) which^depleted during incubation
(Bueding, 1950).In contrast with most other parasitic helminths, adult
schistosomes have been shown to produce only lactate under aerobic and anaerobic conditions (Bueding, 1950; Schiller et al, 1975).
Certain nematodes e.g. Chandlerella hawkinqi and Setaria cervi have a similar metabolism (Barrett, 1981) but no other trematode
appears to exhibit this feature.
The main area of debate regarding energy synthesis in the
adult parasite concerns the extent to which oxidative processes participate in adenosine -5*-triphosphate (ATP) generation.
Bueding and co-workers (1950, 1982) have shown that L-lactate is the major end-product of glucose catabolism in S.mansoni. They also failed to demonstrate a Pasteur effect and so have concluded
that anaerobic glycolysis (Fig.2) is the sole mechanism for
energy production in the parasite. Therefore, schistosomes are
frequently referred to as "homolactic fermenters".
In view of the oxygen tension of blood (49-66 mm Hg, Smyth,
1976), some oxygen would presumably be available to the parasites.
It would be physiologically advantageous for the worms to be able
to harness any available oxygen for more efficient means of energy production. All helminths studied so far consume oxygen
when available (Barrett, 1976a) and S.mansoni is no exception
(Bueding, 1950; Schiller et al, 1975), The exact physiological
role of the oxygen remains to be elucidated, Schillex et al (1975)
oogenesis. Large numbers of mitochondria have been found in
association with the reproductive tissues of female S.mansoni and
5,haematobium (Erasmus, 1973; Burden & Ubelaker, 1981), There
fore, oxidative phosphorylation may be of great significance in the reproductive biology of the parasite. Coles (1972 b, 1973a)
suggested that oxygen is used to generate ATP via an electron transport chain. From incubation experiments, Bueding & Fisher
(1982) maintain that no oxidative processes contribute to energy
formation in schistosomes because of the exact balance obtained
between glucose depletion and lactate production. Additionally,
Bueding & Charms (1952) failed to find sufficient quantities of
cytochromes to account for more than one tenth of the oxygen taken
up by the worms.
More evidence does exist regarding oxidative phosphorylation
in S.japonicum in that Huang (1980) has confirmed the presence of
a succinate - cytochrome C reductase, cytochrome C oxidase,
riboflavin and an ubiquinone - like substance. There have also
been reports of tricarboxylic acid (TCA) cycle (Fig.3) activity in S.mansoni and S.japonicum (Coles, 1973b; Smith & Brown, 1977a;
Huang, 1980). liiaitz (1964) also suggests that schistosomes are capable of operating a hexosemonophosphate (HMP) shunt (Fig.4) and
the first two enzymes of the pathway have been found in S.mansoni
(Smith & Brown, 1970, 1977; Coles, 1973b).
have oxygen is used in the tanning processes during
Figure 2
The glycolytic pathway.
Glucose
ATP AOP
Glycogen
Pho&phoryfaSQ
Glucose-1-P
Phosphogiucomufosa
Glucose-6-P
Glucota-phosphata isomarasa
Fructose-6-P ATP----J
PhotphofructoklnasaAOP
Fructose-1.6-bisP
AidoiaM
Dihydroxyacetone-P Tfiow-*'*orr»rs»«Glyceraldehyde-3-PMAO*--vL„ J G
H* + NAOH 4. — A Glycaraidaftyda-3-P danvdroganaaa
2x1.3-OiphosphoglycerateAOP----S
1 Phoapfioglycarata kmasa ATP A — -A,
2 x 3-Phosphoglycerate
Phosphoglycaromutasa
2 x 2-Phosphoglycerate
FhoiaaaHjO4
Pyruvafa k/naaa
2xPhosphoenolpyruvateaop----JA T P + — A
2 x Pyruvate H* • NAOH--- \J
Lactata dahydroganaiaNAO*
2 x Lactate
*
*
Figure 3.
The tricarboxylic acid cycle.
•f
%
0
*
Pyruvate
Oxaloacetate-----------
NADH + H *Maiatedehydrogenase
r^ — N A D *
Malate
Fumarase
H20
Fumarate
Succinatedehydrogenase
i
r
fpH2
fp
Succinate
i ;Citrate synthase
Citrate
CoAAconitase
Isocitrate
Isocitratedehydrogenase
Oxalosuccinate
c o 2
2-Oxoglutarate
Succinyl-CoA
Figure 4.
The hexosemonophosphate shunt.
GlucoseATP
MAOP NAOPH+H' ADP
Glucose-6-P « 6-Phosphogluconate
Schistosomes ingest large quantities of red blood cells
(Chappell, 1980), From the quantities of haematin visible in the
parasites it is evident that the females ingest more erythrocytesthan the males. Adult erythrocytes are primarily lactate
producers but younger cells are capable of oxidative metabolism
(Harris & Kellermeyer, 1967). Therefore, if erythrocyte enzymes
are active within the parasite gut then assays will include host
enzyme activity. The extent of contamination will depend on the<*l
proportion and development^status of the erythrocyte material in
the schistosome gut. Timms & Bueding (1959) and Coles (1970b)
suggest that erythrocyte enzymes will be denatured in the gut but
Smith & Brown (1970) reported that host enzymes are still func
tional after ingestion. However, this aspect has been largely
neglected by other researchers.
1.2.2. Aims
The present study attempts to evaluate the relative con
tributions made by glycolysis, TCA and HMP pathways to overall
energy metabolism in schistosomes, particularly S.mansoni. This
evaluation is based on the results of in vitro incubation
experiments, intermediary metabolite and specific enzyme analyses.
The study also examines the role of pyruvate kinase in glycolytic
regulation as Brazier & Jaffe (1973) suggested that the enzyme
is not allosteric. Additionally, S.mansoni!s potential foroxidative phosphorylation is investigated
Due to problems of supply and life cycle maintenance, many previous studies have been restricted to S.mansoni, S.haematobium
and S.japonicum. Consequently there is a paucity of comparative
interspecific data relating to these and other species. During
the course of this study, small quantities of schistosomes other than S.mansoni, became available intermittently. Hence it was
possible to extend the range of certain areas of study to include
the following species, S.marqrebowiei, S.japonicum, 5.haematobium,
S.bovis, S.leiperi, S.intercalatum and Schistosomatium douthitti.
Further information relating to schistosomes of primarily veterinary
significance may prove valuable as there have been reports of
S.bovis (Blair, 1966) and 5.marqrebowiei (Walkiers, 1928; Lapierre
& Hein, 1973) infections in man.
Whilst much of the previously published work on schistosome
biochemistry has been concerned with paired worms, the present study is concerned with the separate examination of male and
female metabolism. Also this study attempts to quantify the effect
of schistosome gut contents on specific enzyme analyses.
CHAPTER 2
MATERIALS AND METHODS
2.1. MAINTENANCE
2.1.1. Snails
Uninfected, albino Biomphalaria qlabrata were reared in
dechlorinated tap water, in covered plastic trays (50 x 20 x 4 cm)
at 25°C. Snails were fed with washed lettuce and small quantities
qf commercial fish food (e.g. Tetramin) and the trays were cleaned
once every two weeks. Infected snails were maintained in a similar
fashion except that the snails were isolated in a special bio
hazard area and fed on boiled lettuce only. Bulinus tropicus were maintained in the same manner except that the snails were fed with
pre-soaked and dried sycamore leaves.
2.1.2. Mice
Outbred LACA and To mice of both sexes were used to harbour
S.mansoni and S.marqrebowiei infections respectively. The animals
were maintained under standard animal house conditions.
2.1.3. INFECTIONS
2.1.3.1.Egg collection, hatching and collection of miracidia
Livers and small intestines of mice of greater than six weeks
past-infection were homogenised in approximately 30 ml of 0«9j
(w/v) saline in an MSE blender set at maximum speed for 7 min.The slurry was washed through 500, 300 and 200 mesh sieves with
several volumes of saline. The filtrate was left to stand for
5 min*in a large crystallising dish, after which it was decanted
slowly, leaving the eggs in a small volume of saline. The eggs
were then washed into a smaller glass dish with saline and left to
stand for a further 5 min. Most of the saline was then withdrawn
using a Pasteur pipette and several ml of aquarium water were
added. The dish was then placed near a 15 w bench lamp and
examined under a dissecting microscope after 5 min. Miracidia
were collected using a finely drawn Pasteur pipette and used to infect the appropriate molluscan host.
2.1.3.2. Snail infections
Miracidia of S.mansoni and S.marqrebowiei were used to infect
S.qlabrata and B.tropicus respectively. Individual snails were placed in separate wells containing approximately 5 ml of de-
chlorinated tap water in plastic trays. B.qlabrata were infected
with 4 - 6 miracidia each and B.tropicus with 2 miracidia each.
The trays were then covered and left at room temperature for
between 12 - 24 h. The snails were then transferred to an isolated
biohazard area and maintained as described previously.
2.1.3.3. Collection of cercariae
Snails were examined at 35 days post infection (DPI) for
cercarial production (shedding). Small glass jars containing
about 50 ml of dechlorinated tap water were used to hold 1 0 - 1 5
infected snails each. The jars were placed as close as possible
to a 15 w bench lamp and examined at 15 min intervals. Cercariae were collected using a finely drawn Pasteur pipette. All
procedures involving collection and handling of cercariae were
carried out in an isolated biohazard area whilst observing the
utmost safety precautions.
On occasions, cercariae of S.bovis, S.leiperi, 5.haematobium
S.marqebowiei, S.douthitti and 5..japonicum were obtained from the
British Museum of Natural History and Winches Farm field station,
cercariae were transported to the department in sealed 20 ml
plastic vials enclosed in four layers of polythene. On arrival,
the cercariae were taken to the biohazard room and treated as above
2.1.3.4. Mouse infections
Infection procedures were based on the method of Smithers
& Terry (1965). Mice older than 5 weeks were used to harbour
adult parasites. The animals were anaesthetised by intraperitoneal injection of sodium pentabarbitone (Sagatal, May & Baker, Ltd)
diluted 1:9 with sterile distilled water at a dosage of 0*1 ml/g body weight. This level generally maintained anaesthesia for
between i - 1 h. 16 mm, 25 G hypodermic syringes were used to administer the drug.
As much hair as possible was removed from the ventral abdominal region of the mice using sharp scissors. The animals
23
were then placed in rows, on their backs, between close fitting
slats of wood. Depilated areas were moistened with dechlorinated
water and metal rings (1 cm internal dia x 0*5 cm high) were applied. Cercariae were placed in the lumen of each ring in as small a volume as practicable. The routine infection level was approximately
200 cercariae per mouse. The animals were left for at least 30 min
after which the rings were removed and the mice returned to their
cages. All working surfaces were then doused with copious amounts
of absolute alcohol and left overnight.
2.1.4. RECOVERY OF ADULT PARASITES
Techniques for the recovery of adult parasites were based on
the method of Yolles et al (1947) and Duvall & De bJitt (1967).
Animals were killed by placing them in a chloroform jar. A circumferential dermal incision was made at approximately the stomach region and the fore and hind areas of skin were pulled away to
expose as much of the abdomen as possible. The ventral body wall
was cut away to expose the viscera and part of the rib cage was
removed to allow access to the dorsal aorta. The hepatic portal vessel was severed as close to the liver as possible and the
visceral organs displaced over the lip of a glass funnel. A 25 mm
19 G hypodermic syringe was inserted into the dorsal aorta and used to inject 15 - 20 ml of saline (0»9% w/v Na Cl) or citrated
saline (5 mg sodium citrate/ml saline). The perfusate was
allowed to run into a collecting dish through a nylon fine meshtea strainer
The funnel was washed through with several ml of saline
and perfused worms were removed from the strainer with fine forceps
and placed in a petri dish containing 0*9? (w/v) saline. The
visceral surfaces and mesenteric vessels were examined and any remaining parasites were removed using fine forceps and transferred
to the saline pending further analysis.
2.2. CHEMICALS
All enzymes and other reagents were obtained from Sigma or
Boehringer Mannheim unless otherwise stated.
2.3. CARBOHYDRATE AND WET WEIGHT DETERMINATIONS
Glucose and glycogen levels in male and Pemale S.mansoni and
S.marqrebowiei were measured at 42, 56, 70 and 84 days post infection.
Worms were removed as described in section 2.1.4, separated with
fine forceps and rinsed three times in ice-cold 0-9$ (w/v) saline.
Batches of 5 male and 8 female parasites were blotted dry on tissue2paper and placed onca pre-weighed 1 cm stainless steel grid. A
hot air drier was held for 10 sec at a distance of about 15 cm to
remove additional surface water. The worms were then weighed on a Mettler analytical balance accurate to 0*1 mg. The parasites
were then removed and placed in 100 |jl of ice-cold 0«6N perchloric
acid in pre-cooled, glass, 0*1 ml mini-homogenisers (Jencons)
and disrupted with approximately 60 passes of the pestle.
Homogenates were then transferred to 1*5 ml Eppendorf tubes
and left at 4°C for 45 min. The tubes were then centrifuged at
14000 g for 10 min in a Jobling mini-centrifuge and the supernatants
used for glucose and glycogen determinations according to the
method of Keppler & Decker (1974) (section 2.8.1,2.). The centri
fuged pellets were assayed for total protein using a modified
method of Loiury et al (1951) (section 2.4.).
2.4. PROTEIN DETERMINATIONS
Protein measurements tuere performed on perchlorate pre
cipitated pellets using a modified method of Lowry et al (1951). Constituent reagents were reduced proportionately so that the final
assay v/olume in each tube was 3 ml. Protein pellets were re
suspended in 0*5 ml of 0*5 M NaOH and heated at approximately 80°C in a water bath until they dissolved. Bovine serum albumen
(BSA) was made up to 1 mg/ml with 0«5 M NaOH and heated with the
samples. Standards (20, 50, 100 & 200 pg protein) and samples
were made up to 0«8 ml with 0*5 M NaOH in-separate ts3t -tubes.
2 ml of Lowry reagent was added-to each sample and standard and
the-tubes were left for 10 min at room temperature. 0-2 ml of Folin - Ciocalteau reagent were added to each tube and readings were taken 30 min later on a Cecil 202 Spectrophotometer at
750 nm. A comparison was made between this method and the Bradford dye binding technique (1976) using BSA standards.
2.5. LIPIDS
Total lipid levels in male and female S.mansoni were measured
at 42, 50, 70 and 84 days post infection. Adult parasites were
obtained and washed as described in section 2.1.4. 5 - 10 malesand 1 0 - 2 0 females were homogenised in 100 |jl of a chloroform :
methanol (2:1) mixture in glass, 0*1 ml, mini-homogenisers (Jencons)
with about 60 passes of the pestle. The homogenates were transferred to 1*5 ml Eppendorf tubes and left overnight at 4°C. The tubes
were then centrifuged in a Jobling mini-centrifuge for 5 min (14000 g) and the supernatants were removed and stored at 4°C. The pellets
were re-suspended in 100 jjl of chloroform/methanol and left at 4°C
for 60 min. The tubes were centrifuged for 5 min and the supernatants
combined with the respective previous fractions in separate tubes.
The pellets were re-suspended in 100 il each of ice-cold 0-6 N
perchloric acid and left at 4°C for 45 min. The pellet material
was then centrifuged for 10 min as above, the supernatants discarded
and the pellets assayed for protein as previously described. The
lipid samples were evaporated to dryness under a stream of nitrogen.
2 ml of concentrated sulphuric acid was added to each test tube and the tubes heated in a boiling water bath for 10 min. The total lipid levels were determined using the method of Zollner & Kirsch
27
*
%
%
A
%
2.6. ENZYME ASSAYS
Enzyme analyses were performed on extracts of adult males and
females of six schistosome species, namely S.mansoni, S.marqrebowiei,
S.bovis, S.leiperi, S..japonicum and 5.haematobium. Worms were removed as previously described and rinsed with three changes of
ice cold 0*9? (w/v) saline. Male and female parasites were separated
and homogenised in pre-chilled, glass mini-homogenisers (Jencons)
using the extraction medium of Zammitt etal^ (1976); 50 mM
Triethanolamine (TRA)/HCL, 1 mM ethylenediamine tetra-acetic acid
(EDTA), 2 mM MgCl2 and 30 mM 2 - mercaptoethanol, at pH 7.5. The
volume of medium was adjusted to give approximately a 1:5, worm : pi
medium ratio. The extraction of phosphofructokinase was performed
using the medium of Opie & Newsholme (1967) ; 50 mM Tcis/HCL,1 mM EDTA and 5 mM MgSO^ at pH 8*2. The homogenates were centrifuged
at 30,000 g for 10 min at 2°C in an M.S.E. High Speed 25 centrifuge and the supernatants taken for enzyme and protein analysis.
Tricarboxylic acid enzyme activities in S.mansoni were assayed
in sonicated and intact homogenates using up to 70 pg of soluble
protein per assay. Ultrasonic disintegration was performed using
an M.S.E. Soniprep 150 using 3 x 10 sec phases of 20 p with 30 sec
intervals, with the sample packed in ice.
Assays were perfomed at 30°C in a total assay volume of 1 ml using a Cecil 505 recording spectrophotometer. All reactions were
followed at 340 nm except where stated, initiated by the addition
of supernatant (except pyruvate kinase) and measured using the following methods:-
The analysis was based on the method of McManus 4 Smyth
(1982).The final reaction mixture contained: 50 mM Phosphate, pH
7»4; 0 1 mM EDTA; 0*5 mM NADP; 10 mg Glycogen; 5 y M Glucose-1,
6 - bisphosphate; 15 mM Mg C^; 2 mM AMP; 71) Phosphoglucomutase;
5U Glucose -6- phosphate dehydrogenase.
Hexokinase (E.C. 2.7.1.1.)The assay was based on the method described in Biochemica
information (Boehringer Mannheim, 1975). The final reaction
mixture contained: 50 mM TRA/HC1, pH 7*4; 200 mM D-Glucose;
10 mM MgC^J 3 mM ATP; 0*7 mM NADP; 5U Glucose -6- phosphate
dehydrogenase.
Glucosephosphate isomerase (E.C. 5.3.1.9.)The assay was based on the method of McManus & Smyth (1982).The final reaction mixture contained: 100 mM TRA/HC1, pH 7»45
2 mM Fructose -6- phosphate; 0*5 mM NADP; 7 mM MgCl^; 5U Glucose
-6- phosphate dehydrogenase.
Phosphoqlucomutase (E.C. 2,7.5.1.)The assay was based on the method of McManus 4 Smyth (1982).
The final reaction mixture contained: 100 mM TRA/HC1, pH 7*4; 5 mM Glucose -1- phosphate; 0*2 mM Glucose - 1, 6 - bisphosphate; 1 inM
EDTA; 0.5 mM NADP; 7 mM MgC^; 5U Glucose -6- phosphate dehydrogenase.
Phosphorylase a + b (E .C . 2 . 4 . 1 . 1 . )
P h osphofructokinase (E . C . 2 . 7 . 1 . 1 1 . )
The assay was based on the method of McManus & Smyth (1982) The final reaction mixture contained: 100 mM TRA/HC1, pH 8*0;
100 mM Ammonium sulphate; 1 mM EDTA; 5 mM Fructose -6- phosphate;
2 mM ATP; 0»1 mM NADH; 5: mM Mg 12U Glucose -3- phosphate
dehydrogenase; 10U Aldolase; 10U Triosephosphate isomerase.
Aldolase (E.C. 4.1.2.13).
The assay was based on the method of McManus & Smyth (1982)
The final reaction mixture contained: 100 mM TRA/HC1, pH 7*4; 0*4
mM Iodoacetate; 5 mM Fructose - 1 , 6 - bisphosphate; 0.1 mM NADH; 12u Glycerol -3- phosphate dehydrogenase; 10U Triosephosphate
isomerase.
Triosephosphate isomerase (E.C. 5,3.1.1.)
The assay was based on the method of McManus & Smyth (1982)
The final reaction mixture contained: 200 mM TRA/HC1, pH 7«4;
5 mM Glyceraldehyde -3- phosphate; 0*1 mM NADH; 12u Glycerol -3-
phosphate dehydrogenase,
Glyceraldehye -3- phosphate dehydrogenase (E.C. 1,2.1.12.)
The assay was based on the method of McManus & Smyth (1982)
The final reaction mixture contained: 100 mM TRA/HC1, pH 7*A;
1 mM ATP; 1mM EDTA; 2 mM Mg SO^; 6 mM Glycerol -3- phosphate;
0*1 mM NADH; 15U. Phosphoglycerate kinase.
Phosphoqlycerate kinase (E.C. 2.7,2.3.)
The assay was based on the method of McManus & Smyth (1982)
The final reaction mixture contained: 100mM TRA/HC1, pH 7*4;1 mM ATP; 6 mM Glycerol -3- phosphate; 1 mM EDTA; 2 mM Mg S0^;□ •1 mM NADH; 12U Glycerol -3- phosphate dehydrogenase.
Phosphoqlycerate mutase (E.C. 2.7.5.3.)
The assay was based on the method of McManus & Smyth (1982)
The final reaction mixture contained: 100 mM TRA/HC1, pH 7*4;
1 mM Mg S0^; 1 mM ADP; 0-1 mM NADH; 5 mM Glycerol -3- phosphate; 0*1 mM 2, 3- Diphosphoglycerate; 10U L-lactate dehydrogenase;
8U Enolase; 10U Pyruvate kinaSe.
Enolase (E.C. 4.2.11.)
The assay was based on the method of McManus & Smyth (1982)
The final reaction mixture contained: 100 mM TRA/HC1, pH 7*4;1 mM Mg S0^; 1 mM ADP; 0*1 mM NADH; 1 mM 2- Phosphoglycerate;10U Lrlactate dehydrogenase; 10U Pyruvate kinase.
Pyruvate k in ase (E . C . 2 . 7 , 1 . 4 0 ).
The assay was based on the method of Brazier & Jaffe (1973).The final reaction mixture contained: 100 mM TRA/HC1, pH 7«4;
5 mM Mg 50^; 40 mM KC1; 5 mM ADP; 5 mM Phosphoenolpyruvate;
0*1 mM NADH; 1QU L-lactate dehydrogenase. The reaction was started
by the addition of Phosphoenolpyruvate.
Lactate dehydrogenase (E.C. 1,1,1.27)
The assay was based on the method of Opie & Neusholme (1967).
The final reaction mixture contained: 50 mM Tris/HCl; pH 7*4;
□•1 mM NADH; 1 mM KCN; 5 mM pyruvate.
Alcohol dehydrogenase (E.C. 1.1.1.1.)
The assay was based on the method shown in Biochemica
information (Boehringer Mannheim, 1975). The final reaction
mixture contained: 75 mM Glycine - Sodium pyrophosphate, pH 9*0;70 mM Semicarbazide; 0-1 ml Ethanol; 1 mM NAD; 10 mM Glutathione.
Pyruvate dehydrogenase (E.C. 1.2.4.1.)
The assay was based on the method of Tai at al (1982) and
the final reaction mixture contained: 200 mM Tris/HCl, pH 8; 20 mM
NAD; 20 mM Acetyl Co enzyme A; 50 mM Dithiothreitol; 500 mM MgC12J
200 mM Thiamine pyrophosphate; 0*5 Arylamine acetyl transferase;
0«5U 4- Aminoazobenzene -4*- sulphonic acid; 10 pi Triton x-100;
500 mM Pyruvate, Activity was determined from the decrease in
optical density at 460 nm.
Citrate synthase (E,C. 4.1.3.7.)
The assay was based on the method of McManus & Smyth (1982) and the final reaction mixture contained: 100 mM Tris/HCl, pH 7*4;
6 mM Malate; 0*2 mM NAD; ; 0»2 mM Acetyl Coenzyme A;
10U Malate dehydrogenase; + 10 pi Triton x-100.
Aconitase (E.C. 4,2.1.3.)
The assay was based on the method of McManus & Smyth (1982).
The final reaction mixture contained: 100 mM Tris/HCl, pH 7*4;
100 mM NaCl; 0*1 mM Cis-aconitate. Activity was measured from the change in absorbance at 240 nm.
Isocitrate dehydrogenase (E.C.1.1.1.41.)
The assay was based on the method of McManus & Smyth (1982).
The final reaction mixture contained: 100 mM Tris/HCl, pH 7*4;
2 mM ADP; 2 mM NAD; 8 mM Mg C ^ or Mn C^j 5 mM DL-isocitrate.
I s o c it r a te dehydrogenase (E . C . 1 . 1 . 1 . 4 2 . )
The assay uias based on the method of McManus & Smyth (1982) and the final reaction mixture contained: 100 mM Tris/HCl, pH 7»4;
0*05 mM NADP; 0*8 mM Mg or Mn 5 mM DL-isocitrate.
2 - oxoqlutarate dehydrogenase (E.C, 1.2.4.2.)
The assay was based on the method of McManus & Smyth (1982). The final reaction mixture contained: 100 mM Phosphate, pH 7*4;
5 mM KCN; 1 mM Fe (CN)g; 5 mM 2 - oxoglutarate. The change in absorbance was measured at 420 nm.
Succinate dehydrogenase (E.C. 1.3,99.1.) (FUM-SUCC)
The assay was based on the method of McManus & Smyth (1982)
and the final reaction mixture contained: 100 mM Phosphate, pH 7»4; 0«05 mM Ca C 2 mM Mg Cl^J 0*1 mM NADH; 30 mM Fumarate. The reaction was measured at 420 nm.
Succinate dehydrogenase (E.C. 1.3.99.1.) (5UCC.-FUM)
The assay was based on the method of McManus & Smyth (1982)
and the final reaction mixture contained: 100 mM Phosphate, pH 7»4; 1 mM i<2 Fe (CN)gj 40 mM Succinate; 1 mM KCN; 1 mM EDTA. The change
in absorbance was followed at 420 nm.
Fumarase (E . C . 4 . 2 . 1 . 2 . )
The assay was based on the method of McManus & Smyth (1982) and the final reaction mixture contained: 100 mM Phosphate, pH 7»4;
50 mM Malate. The change in optical density was followed by 240 nm.
Malate dehydrogenase (E.C. 1.1.1,37.) (HAL- QAA)
The assay was based on the method of McManus & Smyth (1982)
and the final reaction mixture contained: 50 mM Tris/HCl, pH 7«4;1, mM NAD; 1 mM KCN; 1 mM Malate.
Malate dehydrogenase (E.C. 1.1.1.37) (QAA-MAL)
The assay was based on the method of McManus & Smyth (1982) and the final reaction mixture contained: 50 mM Tris/HCl, pH 7*4;0»1 mM NADH; 1 mM KCN; 1 mM Oxaloacetate.
Qctopine dehydrogenase (E.C. 1.5.1.11.)
The assay was based on the method of Storey & Dondo (1982)
and the final reaction mixture contained: 100 mM Imidazole/HCl, pH 7*0; 10 mM L-arginine; 0«1 mM NADH; 1 mM Pyruvate.
Phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32.)
The assay was based on the method of Opie & Newsholme (1967)
and the final reaction mixture contained: 70 mM Tris/HCl, pH 7*4;
1 mM Mn C^; 0*1 mM NADH; 2 mM Inosine -51- diphosphate 15 mM NaHCO^J 10U Malate dehydrogenase.
Glucose -6- phosphate dehydrogenase (E.C, 1,1,49.)
The assay was based on the method of McManus & Smyth (1982)
and the final reaction mixture contained: 100 mM TRA/HC1, pH 7*4;
2 mM Glucose -6- phosphate; 0«5 mM.NADP; 7 mM MgCI^.
6 - Phosphoqluconate dehydrogenase (E.C. 1,1,44.)
The assay was based on the method of McManus & Smyth (1982)
and the final reaction mixture contained: 100 mM TRA/HC1, pH 7*4;
2 mM PHosphogluconate; 0«5 mM NADP; 7 mM Mg
Transaldolase (E.C. 2,2,1,2.)
The assay was based on the method shown in Biochimica Information (Boehringer Mannheim, 1975) and the final reaction
mixture contained: 80 mM TRA/HC3, pH 7*4; 8 mM EDTA; 0*5 mMErythrose -4- phosphate; 7 mM Fructose -6- phosphate; 0«1 mM NADH;
5L) Glycerol -3- phosphate dehydrogenase; 10U Triosephosphateisomerase.
M a lic enzyme ( E . C . 1 . 1 . 1 . 4 0 . ) (MAL-PYR)
The assay was based on the method of McManus & Smyth (1982)
and the final reaction mixture contained: 100 mM Tris/HCl, pH 7-4
7 mil Mg C12; 0*5 mM NADP; 20 mM Malate.
Malic enzyme (E.C. 1.1.1.40.) (PYR-MAL)
The assay was based on the method of McManus & Smyth (1982)
and the final reaction mixture contained: 100 mM Tris/HCl, pH 7*4
50 mM Pyruvate; 80 mM KHCO^; 1 mM Mn Cl^; 0*1 mM NADPH.
Nucleoside - 5 dipfaosphate kinase (E.C. 2.7.4.6.)
The assay was based on the method shown in Biochimica
Information (Boehringer Mannheim, 1975) and the final reaction mixture contained: 80 mM TRA/HC1, pH 7*4; 2*25 mM ATP; 1 mM
Phosphoenolpyruvate; 17 mM Mg C^; 65 mM KC1; 0*1 mM NADH; 10U
Pyruvate kinase; 10U L-lactate dehydrogenase; 1 mM Deoxythymidine -5T- diphosphate.
Glutamate - oxaloacetate transaminase (E.C. 2.6.1.1.)
The assay was based on the method shown in Biochimica Information (Boehringer Mannheim, 1975) and the final reaction
mixture contained; 95 mM Phosphate, pH 7*4; 180 mM L-aspartate;
19 mffl 2 - oxoglutarate; 0*1 mM NADH; 8U malate dehydrogenase.
Enzyme activities are expressed in terms of n moles of product formed/min/mg soluble protein. Protein determinations were performed on 20 - 50 pi aliquots of supernatant as outlined in section 2.4
after precipitation with 20-50 ul of ice-cold 0*6 N perchloric acid and centrifugation at 14000 g for 10 min.
2.7. METABOLITES
*
♦
*
#
sWteSteady^levels of certain intermediary and end-product meta
bolites were measured in both sexes of S.mansoni and S,marqrebowiei
using spectrophotometric and fluorimetric methods of analysis. Com
parisons between these procedures were made using known concentra
tions of standard solutions (100, 120 & 140 nM) and mouse liv/er
material.
2.7.1. SPECTROPHOTOMETRY
2.7.1.1. Preparation of materialj
Parasites were obtained as described in section 2.1.4. rinsed
rapidly in ice-cold 0»9$ (w/v) saline and plunged into liquid nitrogen.
Parasites between 42 - 84 days post infection (DPI) were used for
determinations. Frozen material was rapidly weighed (if sample
was large enough) and either processed immediately or stored in
liquid nitrogen until required. Parasite tissue was powdered in
liquid nitrogen using a porcelain mortar and pestle. Ice-cold 0«6N
perchloric acid was added to the samples in the ratio of approximately 20$ w/v. Continual mixing with the pestle thawed the perchloric acid and the powdered material and the samples were left
for 45 min at 4°C. After this, extracts were transferred to
Eppendorf tubes and centrifuged in a Jobling mini-centrifuge at 14000g for 10 min. The protein pellets were assayed as described
in section 2.4. The supernatants were neutralised by addition of
20 pi aliquots of 3*75 M KHCO^ and then re-centrifuged as before. The supernatants were removed with a Pasteur pipette and used for
assay. Samples of mouse liver were dissected from unifected chloroformed LACA mice. Pieces of liver were plunged into liguid
nitrogen, rapidly weighed and treated as for the parasite material.
2.7.1.2. Assays
All assays (0*5 or 1 ml final volume) were performed on a
Cecil 505 recording spectrophotometer at room temperature using
the techniques described below. Optical density changes were
followed at 340 nm.
Adenosine -51- triphosphate
The assay was based on the method of Jaworek et al (1974) and the final reaction mixture contained: 100 mM Triethanolamine
(TRA)/HC1, pH 7»4; 5 mM 3 - Phosphoglycerate; 3 mM Mg S0^; 1 mM
Ethylenediaminetetra-acetic acid (EDTA); 0*1 mM NADH; 12U
Phosphoglycerate kinase; 10U Glyceraldehyde -3- phosphate dehy
drogenase.
Adenosine -5! - diphosphate . & Adenosine -5!- monophosphate
The assay was based on the method of Jaworek et al (1974)
and the final reaction mixture contained: 200 mM TRA/HC1, pH 7«4;
100 mM l^CO^; 1 mM Phosphoenolpyruvate; 30 mM Mg S0^; 100 mM KCl;
0*1 mM NADH; 10U L-lactate dehydrogenase; 10U Pyruvate kinase; 15U
Myokinase.
Fructose - 1 , 6 - bisphosphate
The assay was based on the method of Mschal et al (1974) and the final reaction mixture contained; 200 mM TRA/HC1, pH 7*6; 0*1
mM NADH; 10U Glyceraldehyde -3- phosphate dehydrogenase; 10U
Triosephosphate isomerase; 10U Aldolase.
Oxaloacetate
As described in section 2.8.1.1.
Phospoenolpyruvate> Pyruvate & 2-Phosphoqlycerate
The assay was based on the method of Czok & Lamprecht (1974) and the final reaction mixture contained; 300 mM TRA/HC1, pH 7*4; 0-1 mM NADH; 20U L-lactate dehydrogenase; 1 mM ADP; 10 mM Mg S0^;
35 mM KC1; 10U Pyruvate kinase; 20U Enolase.
2.7.2. FLU0RIMETRY
2.7.2.1. Preparation of material
Parasite and mouse liver samples were prepared as for the spectrophotometric analysis except that the final, neutralised
sample was filtered across a Gelmann Acrodisc 0*2 |j filter (FLOlii) in order to exclude as much dust as possible.
2,7.2.2. Assays
Metabolites were determined using standard enzymatic tech
niques and the serial addition of linking enzymes and co-factors
enabled several metabolites to be determined in a single sample.
Oxidation of NADH was measured on a Perkin-Elmer 2000 fluorescence
spectrophotometer (excitation wavelength 340 nm, emission wave
length 464 nm). The initial assay volume was 2*2 ml and contained between 0*1 - 0*5 ml of neutral perchlorate extract. The order of
addition of co-factors and enzymes (Table 1) was based on the work
of Colson & Klapper (1979) and Barrett & Butterworth (1982).
Known amounts of metabolites ( 5 - 1 0 nmoles) were added as internal
standards after each determination. Concentrations of metabolites
in samples were calculated from the relative changes in fluores
cence caused by endogenous metabolites and their respective
standards. Linking enzymes were also checked for inherent
fluorescence by adding a further aliquot after each metabolite
measurement to determine if there were any subsequent changes in
fluorescence. The increase in volume after each individual add
ition to the cuvette has to be taken into account for the calculations.
A control cuvette, using triple distilled water in place of
the sample, was run with each assay. Fluorescence readings were taken at 30 sec intervals for 5 - 1 0 min. The data was subsequently
fitted with regression lines and the straight line portions of the
plots were used to calculate changes in fluorescence.
Table 1
The sequential assay of ten metabolites in S.mansoni and
S.marqreboujiei by fluorimetry.
Metabolite determined Enzyme and/or co-factors added
Oxaloacetate Malate dehydrogenase (30 U/ml)
Dihydroxyacetonephosphate Glycerol-3-phosphate dehydrogenase (9U/ml)
Glyceraldehyde-3-phosphate Triosephosphate isomerase (10 U/ml)
Fructose-1, 6-bisphosphate Aldolase (3 U/ml)
2-0xoglutarate NADH (0*03 mM), Aspartate (0*4 mM) Glutamate - oxaloacetate transaminase (6 U/ml)
Pyruvate L-lactate dehydrogenase (21 U/ml)
Phoshoenolpyruvate ADP (0*2 mM), Pyruvate kinase (3 U/ml)
2-Phosphoglycerate Enolase (2 U/ml)
3-Phosphoglycerate 2, 3-Diphosphoglycerate (1 uM) Phosphoglyceromutase (11 U/ml)
Isocitrate Pyruvate (0*02 mM), MnCI- (0*1 mM), NADP (0*5 mM), Isocitrate dehydro- • genase (1 U/ml)
The reaction mixture initially contained; 100 mM TRA/HC1, pH 7*5;
0*02 mM NADH; 2 mM Mg C12; AO mM KOI; 0*1 - 0*5 ml sample. The figures in parentheses-represent the concentrations of enzyme/co- factor at the particular stage of the assay.
All data from the spectrophotometric and fluorimetric assays
of parasite and mouse liver material are expressed in terms of
nmoles/g wet weight. If a particular batch of material was too small to be weighed accurately, the protein value obtained was
extrapolated from the protein; wet weight ratios in section 3.6.
2.8. INCUBATIONS
6h incubations were performed under sterile conditions with
single males, females and paired S.mansoni and 5.marqrebowiei, single
males and females of S.haematobium (Morrocan strain) and males of
S.douthitti.
The incubation medium consisted of Earle's balanced salt
solution (DIFCO), containing 10$ inactivated newborn calf serum
(GIBC0BI0CULT) with 1 /ml of a penicillin/streptomycin mixture
(1000 /1000 ug per ml respectively)(FLOlii). All incubations were carried out in autoclaved 1*5 ml Eppendorf tubes using a maximum
volume of 1*2 ml of medium in each tube. The adult worms were
obtained as described in section 2.1.4. and all subsequent operations involving the transfer of parasites from their host to
the incubation tubes were carried out in a Microflow lamina flow cabinet. All instruments used throughout the operation were sterilised by boiling for at least 15 min.
Earle's solution was made up with sterile triple distilled
water and the antibiotics added prior to filter sterilisation
across a 0*2 |j filter using a vacuum pump. This solution was made up the day before each experiment and stored at 4°C until
required. The correct volume of serum was added under sterile
conditions before each incubation. An aliquot was decanted for pH measurement and the complete medium was pre-incubated in a water
bath at 37°C. The blood glucose levels in the host mice had
previously been measured as 0*88 ± 0-002 mg/ml (4.9 mM) using the
method of Keppler & Decker (1974). Therefore, the glucose concen
tration in the medium was accordingly adjusted to 5 mM by addition
of an appropriate volume of sterile triple distilled water prior
to filter sterilisation. The same batches of Earle*s salts,
serum and antibiotics were used for all experiments to ensure consistent incubation conditions. The ratio of parasites to
incubation medium used was approximately 1 worm: 200 pi medium.
Aliquots of medium were pipetted into Eppendorf tubes, sealed and pre-incubated at 37°C in a water bath. Several petri dishes
containing 10 ml of sterile medium each were maintained at 37°C
on a thermostatically controlled heating dish within the flow
hood and used for washing the worms. The parasites were removed
from mice, placed in 0*9% (w/v) saline, the majority separated
under a dissecting microscope and transferred to the petri dishes.
The worms were washed with four changes of medium and then divided
into two batches. One batch of worms we s- transferred to Eppendorf tubes and placed in a shaking water bath (at minimal setting) at 37°C. Control tubes containing no worms were run concurrently.
The tubes were inverted once every hour. The remaining worms were
transferred to clean Eppendorf tubes and stored in liquid nitrogen and were subsequently used as unincubated controls.
After incubation, the worms were removed and each batch was
washed in three changes of sterile, ice-cold 0 9 $ (w/v) saline with vortexing on a rotamix (Tucker Ltd) for 10 sec. This was
done to remove incubation medium from the parasites. The worms
were then transferred to clean Eppendorf tubes prior to storage in liquid nitrogen, 50 pi aliquots were removed from each incubate
for pH measurement. Protein in the media samples was precipitated
by adding M % (v/v) of ice-cold 0*6N perchloric acid and maintaining
the samples at 4°C for 1 hour. Each tube was then spun at 14000 g
in a Jobling mini centrifuge for 10 min, the supernatants decanted,
neutralised with solid KHCO^ and then re-centrifuged. The super
natants were pipetted into clean Eppendorf tubes and stored in liquid nitrogen.
2.8.1. END PRODUCT ANALYSIS
2.8.1.1. Medium
Media samples (10 - 50 pi) were assayed at room temperature
for the presence of alanine, acetate, citrate, ethanol, glucose,
glycerol, D & L-lactate, oxaloacetate, pyruvate, propionate and
succinate using a Cecil 505 recording spectrophotometer in a total cuvette volume of either 0*5 ml or 1 ml at 340 nm unless otherwise stated. The assay methods used were as follows:-
AlanineThe assay was based on the method of Grassl (1974) and the
final reaction mixture contained: 92 mH Tris/HCl, pH 7*4; 7 mH
2-oxoglutarate; 0«1 mH NADH; 6 U L-lactate dehydrogenase; 10 U
Glutamate-pyruvate transaminase.
Acetate
The assay was based on the method of Bergmeyer & Hollering(1974) and the final reaction mixture contained: 140 mH Triethano
lamine (TRA/HC1, pH 8-4; 30 mH Halate; 10 mH HgC12; 1 mH NAD;0*2 mH Coenzyme A; 3 mH ATP; 20 U Halate dehydrogenase; 5 U Citrate
synthase.
Citrate
The assay was based on the method of Hollering & Gruber (1966)
and the final reaction mixture contained: 200 mH Glycyglycine, pH
7*8; 0«1 mH ZnCl; 0*1 mH NADH; 10 U Halate dehydrogenase; 10 U
L-lactate dehydrogenase; 2 U Citrate lyase.
Ethanol
The assay was based on the method of Bernt & Gutmann (1974) and the final reaction mixture contained: 60 mH Phosphate, pH 8*5;
60 mH Semicarbazide; 15 mH Glycine; 0*5 mH NAD; 20 U Alcohol dehydro
genase.
Glucose
4 mM MgSO^; 1 mM ATP; 1 mM NADP; 5U Glucose -6- phosphate dehydrogenase; 15U Hexokinase.
Glycerol
The assay was based on the method of Eggstein & Kuhlmann(1974) and the final reaction mixture contained: 0*25.mM Glycyl-
glycine, pH 7*5; 3 mM MgSO^ 0*1 mM NADH; 1 mM Phosphoenolpyruvate;
10U Pyruvate kinase; 10U L-lactate dehydrogenase? 5U Glycerokinase.
D or L-Lactate
The assay uias based on the method o f K epp ler & Decker (1974)
and the f i n a l re a c t io n m ixture con ta in ed : 200 mM TRA/HC1, pH 7*4;
The assay was based on the method of Noll (1974) and the final
reaction mixture contained: 0*22 mM Glycylglycine, pH 10; 35 mM
L-glutamic acid; 3 mM NAD; 12U Glutamate-pyruvate transaminase;20U D or L-lactate dehydrogenase.
Oxaloacetate
The assay was based on the method of liJahlefeld (1974) and the
final reaction mixture contained: 133 mM TRA/HC1, pH 7*6; 10 mM
Ethylene diaminetetra-acetic acid (EDTA); 0*1 mM NADH; 5ll Malate
48
4
4
4
4
4
dehydrogenase.
Pyruvate
The assay was based on the method of Czok & Lamprecht (1974)
and the final reaction mixture contained: 200 mM TRA/HC1, pH 7*4;
0*1 mM NADH; 7 mM EDTA; 20U L-lactate dehydrogenase.
Propionate
The assay was based on the method of Holz & Bergmeyer (1974)
and the final reaction mixture contained: 50 mM TRA/HC1, pH 7*4:
20 mM MgC^J 15 mM ATP; 0*8 mM Hydroxylamine; 1 mM Acetate; 20U Acetate kinase; 0*1 M Trichloroacetic acid; 10 mM FeCl 3. The
reaction was followed at 492 nm. Acetate kinase catalyses the phosphorylation of acetate and propionate, therefore it is necessary to perform a separate specific assay for acetate (described pre
viously) to determine propionate levels.
Succinate
The assay was based on the procedure described in Methods
of Enzymatic Food Analysis (1977) and the final reaction mixture
contained: 100 mM Glycylglycine, pH 8»5; 15 mM Mg S0^, 0»1 mM NADH;
0.5 mM Coenzyme A; imM Inosine-51- triphosphate; 1mM Phosphoenol pyruvate; 10LI Pyruvate kinase; 10U L-lactate dehydrogenase; 1 U Succiny1-Coenzyme A synthetase (Succinate thiokinase).
2 . 8 . 1 . 2 . Endogenous m etab o lite le v e ls
Incubated and control (i.e. unincubated) parasites were homo
genised by hand in ice-cold 0»6N perchloric acid in pre-cooled glass
mini-homogenisers (Jencons) at a ratio of approximately 1 worm: 20- 30 pi acid. Homogenates were transferred to Eppendorf tubes and
left at 4°C for 1 hour. Samples were then centrifuged in a Jobling
mini-centrifuge1-.(14000 g) for 10 min. The supernatants were decanted
and neutralised with solid KHCO^,
The centrifuged pellets were assayed for protein as described
in section 2.4 and the neutralised supernatants were re-centrifuged at 14ooo g in a Jobling mini-centrifuge. The supernatants were
subjected to similar metabolite analysis as described for the incu
bation media samples. No analyses were carried out for ethanol,
citrate, oxaloacetate, propionate or pyruvate due to lack of material
Glycogen levels were determined using the method of Keppler &
Decker (1974). This technique entails the enzymatic digestion of
glycogen by amyloglucosidase (AGS) and determination of the resul
ting total hexose levels. Approximately 0*05 ml of worm samples were incubated with 0*05 ml of 1M KHCO^ and 0*4 ml AGS (0*4 mg protein) each, in stoppered Eppendorf tubes in a shaking water bath
(minimal setting) at 40°C for 2 h. 0*3 ml of 0*6Aperchloric acid
was then added to each tube and the samples were subsequently
neutralised with solid KHCO^ and centrifuged at 14000 g for 10 min in a Jobling.mini-centrifuge. The supernatants were then removed and used for assay. Glycogen levels were obtained after the sub-
traction of free glucose levels in undigested samples. Frozen
media and parasite samples were thawed prior to metabolite analysis,
which was performed on the day of thawing.
142.8.1.3. CCU Measurement
14The evolution of CC^ by S.mansoni, S.marqrebowiei and14S.douthitti was measured using D - U - C glucose (250 mCi/mmol)
(Amersham Radiochemicals). The radiolabelled glucose was added to
the Earle*s solution prior to filter sterilisation and the final
concentration in the assay medium was adjusted to 0*5 p Ci/p mol
glucose. Modified Eppendorf tubes were used for this series of
incubations. Two Durham tubes with internal measurements 3 mm diax 9 mm long were fixed with Araldite into each Eppendorf tube at
a position to allow the cap to be sealed. Incubation procedures
were carried out as previously described except that 50 pi of
filter sterilised 0 3 M NaOH was placed into each Durham tube to 14collect any CO2 and the maximum volume of incubate per tube was
□•5 ml. Controls for the experiment consisted of intact tubes (with
medium and NaOH) without worms and intact tubes containing 40 and
20 pi aliquots of mouse blood. The latter was used to assess the14contribution, if any, of mouse blood to CO2 evolution. The blood was
removed before perfusion using a sterile hypodermic syringe and the aliquots quickly pipetted into the control tubes.
After incubation, 0*4 ml of absolute alcohol was added to each
tube by injection using a 16 mm 24 G hypodermic syringe. The puncture
holes were rapidly closed with molten sealing wax and the tubes were
left to equilibrate overnight at room temperature. The worms were
then removed, washed several times in 0*9? (w/v) saline and assayed
for protein as described in section 2.4. The NaOH samples were
removed and subjected to scintillation counting.Determination of radioactivity in the NaOH samples were per
formed using a Tracerlab Spotmatic counter. 100 pi samples and
standards (volumes equivalent to 1000, 5000 and 10,000 disinte
grations per minute (d.p.m,)) were mixed with 1 ml each of Beckman
Ready-Solv scintillation fluid (Beckman Instruments) in 1 ml plastic
inserts. The inserts were capped and each placed in a 20 ml glass
scintillation vial. Counting was performed with 1 cycle of 3
phases of 3 min for each tube. Prior to measuring samples and standards, the background count for eqch insert and vial was deter-
^2.^0- ioctO cJp/^ C '* < lvxoos-ft}
mined. Standards/ywere run with every batch of samples to assess
counting efficiency.
2.8.2. Glucose Incorporation
Tissue incorporation of D - u - glucose by S.mansoni
was measured by incubating worms as described for the ^ C 02
evolution experiments using unmodified Eppendorf tubes. The level
of radioactivity used for this investigation was raised to 1 p Ci/ pmol glucose. After 6 h the worms were removed and washed with
three changes of sterile ice-cold 0 9 % (w/v) saline. The worms were then homogenised by hand in 70% ethanol (1 worm/50pl) using
52
glass mini-homogenisers (Jencons). The homogenates were then trans
ferred to Eppendorf tubes and left overnight at 4°C. The tubes
were centrifuged for 10 min in a Jobling min-centrifuge (14000 g)
and the supernatants discarded. The pellets were suspended in 70$
ethanol (50 pl/worm) and left for 1h at 4°C. The tubes were re- centrifuged as before and the supernatants discarded. The pellets
were re-suspended in 0*5 ml of 0-5 M NaOH each and then heated in a water bath at 50°C until they dissolved, 100 pi aliquots of the solutions
were taken for scintillation counting and the remainder used for
protein determination. The controls used for the scintillation
counting consisted of 100 pi aliquots of 0*5M NaOH minus digested
worm material.The results of the incubation experiments are expressed in
14terms of p moles or nmoles per mg total protein# .CO2 production is expressed in terms of glucose consumption attributable to tricarboxylic acid cycle operation. As the mechanism(s) of glucose
incorporation into parasite tissues is not known, the number of counts per minute as measured by the scintillation counter, were
related directly to the initial glucose concentration in the medium.
2.9. PYRUVATE KINASE KINETICS
The activity of pyruvate kinase (PK) from both sexes of S.mansoni, S.marqrebowiei, S.jeponicunyand male S.bovis was
determined at phosphoenol pyruvate concentrations[PEFj between 0*01 - 5 mM, using the assay outlined in section 2.6. The effects of fructose -1, 6- bisphosphate (FBP) and adenosine -5’- triphospate
(ATP) on enzyme activity were examined separately in both sexes
of S.mansoni and S.marqrebowiei using the following variations in
substrate and modulator concentrations:-
- ATP + FBP (0*001 - 0*125 mM); 0*1 mM PEP + ATP - FBP (Inf'll, 1 pM & 1 m); 1*0 mM PEP
2.9.1. "PHYSIOLOGICAL" ENZYME ACTIVITY
Using the data obtained from the metabolite determinations
(section 3.9.2.1.) PK activity in both sexes of S.mansoni was
assayed at "physiological" levels of phosphoenolpyruvate (PEP)
and adenosine -5*- diphosphate (ADP). In addition, the effects of
"physiological" levels of ATP and FBP on enzyme activity under these conditions were examined individually and jointly using the following methods:-
2.9.1.1. Male enzyme
The reaction mixture contained: 100 mM Triethanolamine
(TRA)/HC1, pH 7*4; 5 mM MgSO^; 40 mM KC1; 0*5 mM ADP; 0*1 mM NADH;Vw
10-50 pM PEP; ± 23 pM FBP; ± 2*25 mM ATP; 20IJ L-lactate deyhfdro- genase.
2.9.1.2. Female enzyme
The final reaction mixture contained: 100 mM TRA/HC1, pH
7*4; 5 mM MgS04; 40 mM KC1; 0*4 mM ADP; 0*1 mM NADH; 10 - 50 pM
PEP; ± 50 pM FBP; 0-6 mM ATP; 20U L-lactate dehydrogenase
2.9.2. Effects of Mg /Mn on enzyme activity and enzyme stability
The effect of 5 mM MiJ'and Mi*f on PK activity of both sexes of
S.mansoni was examined using the "physiological” regimes previously
described in the absence of ATP and FBP. The stability of PK from male and female S.mansoni was determined as previously described in the absence of ATP or FBP. Enzyme activity was measured as
soon as possible following centrifugation of the homogenates and
subsequently after 2, 4 and 7 h.
2.9.3. Host enzyme activity in parasite gut contents
Gut contents of female S.mansoni were obtained by cutting the parasites at approximately half-way along their bodies and
allowing the digest to diffuse into the normal enzyme extraction buffer (section 2.6.) (10 pl/worm) in an Eppendorf tube at 4°C for about 1h. The buffer containing gut contents was decanted and
centrifuged as described in section 2.6.
Samples were assayed for PK activity according to the method
presented in section 2.6. in the presence of 5 mM PEP. Samples
were also examined for glucose -6- phosphate dehydrogenase, malate dehydrogenase (malate - oxaloacetate) and citrate synthase activity
using the methods outlined in section 2.6. This was done to
assess the relative contributions, if any, of host gylcolytic,
tricarboxylic acid and hexosemonophosphate enzymes to the deter-
mination of parasite enzyme activity
2.9.4. House erythrocyte and serum pyruvate kinase
For the purposes of comparison, PK from mouse erythrocytes and serum was examined using the assay shown in section 2.6. Nice
were killed as in section 2.1.4 and blood was obtained from the posterior vena cava of the animals using a 16 mm 25 G hypodermic
syringe. The blood was treated as follows, based on the method of Gutmann & Bernt (1974), House blood was added to a solution
of sodium citrate to give a final concentration of approximately
1 mg citrate/ml sample. For a whole blood preparation, 0«5 ml of
citrated blood was added to 5 ml of ice-cold triple distilled
water and left to stand for 15 min at 4°C to complete haemolysis.
Prior to assay, the haemolysate was centrifuged at 3000 g at 4°C
for 10 min and the supernatant taken for determination.
For the separation of erythrocytes and serum, 2 0 0 j j I aliquots of citrated blood were centrifuged as described above and the super
natants (serum fraction) used for assay. The pellets were sus
pended in 1 ml each of ice-cold 0 * 9 % (w/v) saline and then re-
centrifuged. This washing was performed three times and the final
pellets, were suspended in 2 ml each of ice-cold triple distilled
water and left at 4°C for 15 min. The samples were centrifuged as above and the supernatants containing erythrocyte enzymes were assayed. Serum and erythrocyte samples were also examined for
glucose -6- phosphate dehydrogenase, malate dehydrogenase (malate -
oxaloacetate) and citrate synthase activity as outlined in section
2.6 to assess the relative activities of host glycolytic, tri
carboxylic and hexosemonophosphate shunt pathways.♦
*
2.10 ELECTROPHORESIS
Pyruvate kinase (PK) from male and female S.mansoni was
examined using polyacrylamide gel(PAGE), cellulose-acetate, starch
gel and isoelectric focusing techniques. Parasites were obtained
and washed as described in section 2.3 and homogenised in ice-cold 100 md TRA/HC1 buffer at pH 7*4, in a ratio of approximately 2:1
males and 4:1 females, worm/jjl buffer respectively. The samples
were then centrifuged at 20,000 g at 2°C for 10 min in an dSE High
Speed 25 centrifuge and the supernatants taken for examination.
2.10.1. PAGE
Slab gels and buffers were prepared according to the method
of Hames (1981) and the protocols for gel and resevoir buffer pre
paration are shown in Tables 2 and 3 respectively, douse erythrocyte
samples were also examined using this system and the material was obtained as described in section 2.9.4.
The gels were run on an LKB Vertical Electrophoresis system
at 4°C for 8h at 200 V and 14 h at 20V. Gels were then removed and st-ained using the method of Harris & Hopkinson (1976) and the final reaction mixture contained:
0*5d Tris/HCl, pH 7*4; 9 md Phosphoenolpyruvate; 8 md ADP;
0*01 mM NADH; 5 mM Fructose - 1 , 6 - bisphosphate; 20 mM Mg 50^;
67 mM KC1; 15U/fal L-Lactate dehydrogenase.
The reaction mixture was applied to the gels using filter paper overlays and the gels were left at room temperature to
develop. Gels were examined periodically under an ultra violet
(UV) light source after 5 min and the staining patterns were photo
graphed.
2.10.2. STARCH GEL
Parasite and mouse er^hrocyte samples were prepared as
described in section 2.6 and 2.9.4 respectively. Cotton threads
(5 mm long) were dipped into the samples and pressed into pits
in 9«6j, 1 mm thick, starch gels. The gels were run at 4°C on
horizontal apparatus at 200 V, 10-11 mA for 2h using a 0*1 M Tris/
HC1/0«1 M NaH2 P0^ reservoir buffer at pH 7.5.On completion, the gels were stained for PK activity as
previously described.
2.10.3. CELLULOSE-ACETATE
Parasite and mouse erythrocyte samples were prepared as
described above and separated on cellulose-acetate sheets at room temperature on horizontal apparatus at 250 V. The reservoir
buffer was Tris/Barbital (pH 8*6-9*0) at an ionic strength of 0«03
58
*
m
*
ur
♦
The samples were run for 30 min after which, PK patterns were
developed as described above using filter paper overlays and
recorded photographically.
2.10.4. ISOELECTRIC FOCUSING
Homogenates of male and female S.mansoni and whole mouse
blood were prepared as described above in the ratio of 10 worms/50^1 buffer and 15 worms/SOpl buffer respectively. Separation of proteins
was carried out at 4°C using an LKB ampholine polyacrylamide gel
plate, pH range 3*5 - 9*5 on an LKB multiphor with an LKB 2103
power supply. 5, 15 and 20 pi aliquots of each sample were applied cathodally to the gel on small (5 x 10 mm) pieces of filter paper
and 5pl aliquots of haemolysed human blood were also used to act as a marker. The cathode and anode wicks were soaked in 1M sodium
hydroxide and 1 M phosphoric acid respectively. The power pack
was set to deliver 1*4 KV at 30 Id and the pieces of filter paper
were removed after 1h to prevent tailing of proteins.The experiment was stopped after 2i h and the cathode and
anode wicks were removed together with the underlying gel. The pH
gradient was measured on the gel using an Ingold membrane electrode (Pye Unicam) with readings being taken at 1 cm intervals from the
cathodic end of the gel. The gel was then placed on a clean glass plate and stained for PK as described previously. The gel was left to develop at room temperature and the pattern of staining was
visualised and recorded as described above. Subsequently, the out-
Table 2
Protocols for polyacrylamide gel preparation.
A # 4r
Stackinq Gel
Acrylamide/bisacrylamide(37-5:1)
2-5
Stacking gel stock buffer 5-0
Resolving gel stock buffer1*5% Ammonium persulphate 1 -0
Water 11-5
TEWED 0-015
TEWED = , l\l, - tetramethylenediamine
All values are shoun in ml.
Resolving Gel
7-5< 105S
7-5 10
3-75
1*517.25
0-015
3-75
1-5
14-75
0-015
Table 3
Protocols for stacking gel, resolving gel and resevoir buffer
prepration for polyacrylamide gel electrophoresis.
*
Stacking gel buffer : Tris/HCl (pH 6-8); 6 g Tris was dis
solved in AO ml H^O and titrated to pH 6*8 with
iM HC1. Water was added to make up to 100 ml. Resolving gel buffer : Tris/HCl (pH 8-8); 36*3g Tris and
* 48 ml; HC1 were mixed and made up to 100 ml
final volume with water. The solution was titrated
to pH 8*8 with HC1, if necessary,rResevoir buffer : Tris/glycine (pH 8*3); 15g Tris andK
72g glycine were dissolved in and made up to
51 with water.
*
*
lines of the patterns were drawn in with ink and the distance from
the centre of each band to the origin was measured. A graph plot
of these distances against pH readings was fitted with a regression
line and thus used to derive the isoelectric points for each band.
2.11. CYTOCHROMES
Cytochrome spectra were measured separately in male and female S.mansoni, whole and pepsinised mouse blood and a haematin solution.
Parasite samples were obtained as described in section 2.1.4.
Approximately 200 male and female worms were homogenised separately
in 200|j1 of the normal enzyme extraction buffer (section 2.6) at
4°C. 50 jjI and 100 pi aliquots of male and female samples respect
ively, were used for scanning. Whole mouse blood was obtained from
infected animals as described in section 2.9.4 and 100 pi aliquots
were used for the measurement of cytochromes. Spectral scans were also performed on a sample of rat liver to provide a comparison for the parasite material. 0*5 g of rat liver was homogenised in
5 ml of a 225 mM Mannitol/75 mM Sucrose buffer and 200 pi taken for assay.
Differential cytochrome scans were performed on an Aminco D.lii. —2 UV/VIS spectrophotometer at -196°C. Samples were added to
the reaction mixtures to give a final volume of 750 pi at the
following final concentrations Measurement cuvette (reduced)
225 mM Mannitol; 75 mM Sucrose; 5 mM Sodium phosphate; 10 mM
♦
*
0
0
4*
Morpholinopropane sulphonic acid (MOPS); CM mM Ethylenediamine-
tetra-acetic acid; 1Q |-in Rotenone; a feu/ crystals of sodium
dithionite.
Reference cuvette (oxidised)
As for the measurement cuvette, plus 25 pM Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP).
In order to visualise cytochrome b in samples more clearly,10 mM Succinate and 10 pg Antimycin A were added to separate
measurement and reference cuvettes prior to scanning. In addition,
enzymatically digested u/hole mouse blood and haematin samples were
scanned to determine if any intermediary or final breakdown products
of the blood interfered with the parasite cytochrome scans. 10 mg of pepsin were dissolved in 5 ml of in HC1 and the pH was adjusted
to 2*3 by dropwise addition of 4M NaOH. 3ml of the pepsin solution
were added to 1 ml of citrated whole mouse blood in a 20 ml plastic
screw-top tube and subsequently incubated for 30 min at 37°C and 1h at room temperature. Concurrent to this, 5 mg of haematin
were dissolved in 2 ml of 2M NaOH. 50 pi aliquots of the enzyme digest and haematin solution were added separately to 95 pi of
distilled water and their spectra determined against distilled water blanks, at room temperature, using a recording Pye Unicam
SP100 spectrophotometer. After the initial scans were recorded, a few crystals of sodium dithionite were added to each cuvette to
produce reduced conditions and the samples were re-scanned.
2.12. OXYGEN UPTAKE
♦
♦
<6
*
Oxygen uptake measurements mere performed using a YSI Model
53 Oxygen Monitor (Yellow Springs Instrument Co) with a Clark type
electrode, on male and female S.mansoni. The worms were recovered as described in section 2.1,4 and approximately 10 males and 15
females were placed into separate chambers containg 3 ml of the Earle's medium used for the incubation experiments (section 2.8).
The medium was prepared as described in section 2.8 and pre
incubated at 37°C for 5 min prior to assay.The sample: chambers and oxygen probe were thoroughly cleaned
before use to minimise contamination by micro-organisms. The
assay medium was maintained at 37°C by a circulating water jacket
connected to a thermostatically controlled water pump. Measurements were made using a chart recorder over 1 5 - 2 0 min periods.
10 pi aliquots of KCN were added to each sample and control chamber
using a 5 pi Hamilton syringe (Jencons) to give a final concentration of 20 mM. Control values were obtained by measuring any
changes in samples of media without parasites in the presence and
absence of KCN. Worms were removed after assay and washed with 3 *changes of 0*9% (w/v) saline using vigorous manual agitation for
30 sec prior to assaying for protein as described in section 2.4.
CHAPTER 3
r* ftu 4
♦
*
RESULTS
3.1. MAINTENANCE AND COLLECTION OF PARASITES
3.1.1. Molluscan hosts
Maintenance of Biomphalaria qlabrata stocks proved to be
relatively trouble free. Infected animals survived for an average of approximately one month after the onset of cercarial
shedding which began between 28 - 37 days post infection. Great
difficulties were encountered in the maintenance of Bulinus
tropicus colonies. It was generally observed that the mortality rate of the snails increased and egg production decreased after
approximately 3 months. Consequently, it was decided to abandon the colonies and obtain S.marqrebowiei cercariae from the British
Museum qf Natural History.
3.1.2. Infections
Adequate numbers of miracidia were obtained to maintain an infected snail stock. Yet it was noted, that livers and small intestines from animals of between 6-:9 weeks post-infection
yielded greater quantities of viable miracidia. This was probably related to the level of calcification of eggs that would subsequently increase with time.
#•
m
&
3.1.2.1. Recovery of adult parasites
There was no noticeable difference between the numbers of
parasites recovered using normal or citrated saline. The quantity
of S.mansoni recovered from each host was routinely approximately
10? - 20? of the infection dose (about 200 cercariae per mouse). Recovery of other infections was usually much lower (approx
imately 2 - 1 0 worms per host) but occasionally up to 40 worms
per host were obtained.
3.2. STATISTICAL ANALYSES
All experiments yielding data on the variability of one
parameter against serial increments of another were first tested
for linear or logarithmic relationship using the algebraic form of analysis of variance for testing regression goodness of fit as
described by Clarke (1980) (Table 4). Logarithmic relationships
i.e. log Y = a + bx and Y = a +b logx were tested as outlined in Table 4 by substituting the Y or X values by their logarithms
and retaining the corresponding X or Y values in their original
units. The resulting F values were read from tables of F distribution. If no relationship was established, the differences
between means were compared using the following form of Student*s
t - test (from Clarke, 1980):-
(l^+.l^ - 2 ) = X - Y+ S
N
72
2
t
Table 4
Algebraic form of analysis of variance for determiningregression goodness of fit.
SOURCE OF VARIATION
DEGREES OF FREEDOM
SUM OF SQUARES
MEANSQUARES
TEST
REGRESSION 1 SR MR = SR 1
MR = F(1,N-2)MD
DEVIATION FROM LINE
N-2 SD=S-SR MD = SD N-2
TOTAL N-1 S
5 = (1/N) (M.Zx2 - £ x . £ y )SR - (1 /M) (N.Sxv -Sx.Syl2
(1/N) (N.Ex2 - (£x)2)SO = S - SR
N = number of observations MD = deviation mean of squares
S = total sum of squares MR = regression mean of squares
S2 = sample variance x/y = sample meanSD =s deviation sum of squares FE = sum of observationSR = regression sum of squares FE2= sum squares of observation
The resulting values were compared with tables of t distri
bution. The explanation of the symbols is given in Table 4 .
For all statistical tests, the 95$ probability level was chosen
as the threshold of significance. In analyses involving a time course of measurement, if no linear or logarithmic relationship
could be established, overall changes in levels were determined by
comparing individual mean values against each other using the t -
test.
3.3. CARBOHYDRATE DETERMINATION
3.3.1. GLUCOSE LEVELS
Glucose levels in male S.mansoni showed no overall changes from 42 - 84 days except for a decline (P<0*02) at 70 days post
infection (DPI)(Fig 5). Levels of glucose in female S.mansoni
were similar to the males* from 42 - 70 DPI after which they began
to fall (Fig 5). Glucose levels in male S.marqrebowiei at 42,56 and 84 DPI were statistically similar but the levels at 70 DPI were significantly higher (P<0*05) (Fig 7). The levels of
glucose in female S.margrebowiei at each period post infection
were not statistically different (Fig 7). Glucose levels in male worms were significantly lower than in females at 56 and 84 DPI (P«= 0»05).
Glucose levels in male S.mansoni were significantly higher (P<0.001) than in male 5.marqrebowiei at each measurement period
by approximately 50%. Differences between the females of both
species were only found at 42 and 70 DPI when levels in S.mansoni were 40% higher.
3.3.2. GLYCOGEN LEVELS
Glycogen levels in male S.mansoni at 42, 70 and 84 DPI
were similar but the level at 56 DPI cPo-s1 statistically higher (P<0*01) (Fig 6). The levels in female S.mansoni were similar at
each period post infection (Fig 6). The glycogen content of male
S.mansoni was approximately 50% higher than in female worms at each period post infection.
The glycogen content of male S.marqrebowiei was stable from
42 - 70 DPI but the level of 84 DPI showed a marked statistical increase (P«= 0*001) (Fig 8). Levels in female parasites (Fig 8)
were similar at 42 and 56 DPI but a significant increase (P«=Q*05)
was noted at 70 and 84 DPI. Glycogen levels in male S.marqrebowiei
were apprqximately 40% higher than in female worms at each period post infection.
Glycogen levels in male S.mansoni were higher than in maleS.marqrebowiei at 56 DPI by approximately 50%. This situation was
threversed by the 84 day when levels in male S.marqrebowiei were
45% higher than in S.mansoni. The glycogen content of female
S.mansoni was approximately 42% higher than in female S.marqrebowiei at 42 DPI. After this point, there were no statistical differences between the two species.
Figure 5
The values represent the means of 10 determinations ±
standard deviation.
Glucose levels in male and female S.mansoni over a period
from 42 -84 days post infection.
Figure 6
Glycogen levels in male and female S.mansoni over a period
from 42 - 84 days post infection.
The values represent the means of 10 determinations ±
standard deviation
(jg g
lyco
gen
/ m
g pr
otei
n ug
glu
cose
/ m
g pr
otei
n
Fig . 5
Fig jj
Figure 7
The values represent the means of 10 determinations + standard
deviation.
Glucose levels in male and female S.marqreboiijiei over a period
from 42 - 84 -.post infection.
Fioure 8
Glycogen levels in male and female S.marqreboiuiei over a period
from 42 - 84 days post infection.
The values represent the means of 10 determinations + standarddeviation.
Fig -7
* Fig .8
7
3.4. UJET WEIGHT DETERMINATIONS
*
#
©
©
The wet weight of male S.mansoni showed no overall signifi
cant change between 42 - 84 DPI (Fig. 9). A similar situation was
found regarding female S.mansoni (Fig. 9). Male parasites were
approximately 63% heavier than the females at each period post
infection.
The wet weight of male S.margrebowiei showed an overall significant increase from 42 DPI onwards (Fig. 11). Female worms also
increased in wet weight from 42 - 56 DPI after which the level re
mained constant (Fig. 11). The wet weight of male S.marqrebowiei was significantly higher (P«=0*001) than the females’ at each period
post infection by approximately 64%.
The wet weight of male S.margrebowiei was approximately 72% higher than male S.mansoni from 42 - 84 DPI. Female S.marqrebowiei
and S.mansoni showed similar wet weights at 42 DPI, after which,
the former showed a 78% increase over the remainder of the time
course.
3.5. PROTEIN DETERMINATIONS
Protein levels in male S.mansoni showed no statistical change over the 6 week time course (Fig. 10). In contrast, the level in
female parasites showed a significant decline after 56 DPI (Fig. 10).
Up to this point, there was no statistical difference in protein
levels between both sexes, but from 56 DPI onwards, protein in the females fell to about 50% that of the males.
Protein in male S.marqrebowiei showed a significant (P<Q-05) linear decrease with time (Fig. 12). Levels of protein in female
S.marqrebowiei showed no change from 42 - 70 DPI, after which there was a significant decline (P«=0*01) of approximately 40?
(Fig. 12). Protein levels in the male parasites were significantly
(P«= 0*001) higher than in the females by approximately 52?.
Male S.marqrebowiei had higher levels of protein (approximately
63?) than S.mansoni males over 42 - 84 DPI. Female S.mansoni and
S.marqrebowiei showed similar protein levels at 42 DPI but subsequently, the latter were found to have levels approximately 52? higher.
Analysis of the values in Table 5 showed that the Lowry
technique of protein determination was significantly more accurate
(P<0*02) than the Bradford method.
3.6. PROTEIN : WET WEIGHT RATIOS
The ratios in male S.mansoni showed no overall significant
decrease (P< 0*001) between 42 - 84 DPI (Fig. 13). In female
S.mansoni the ratios showed no statistical differences up to 70 DPI (Fig. 13) after which, there was a significant decline (P«=0*001)
of about 45?. The ratios in male S.marqrebowiei decreased significantly (P«=0*001) between 42 - 56 DPI, after which the levels remained constant (Fig. 14). The ratios in female S.marqrebowiei showed an overall decrease with time (Fig. 14).
Figure 9
The values represent the means of 10 determinations ± standard deviation.
liiet weight of male and female S.mansoni over a period from 42 -
84 days post infection.
Figure 10
Protein content of male and female S.mansoni over a period from 42 - 84 days post infection.
The values represent the means of 10 determinations ± standard
deviation.
MS p
rote
in /
wor
m
mg
wet
Wei
ght
/ w
orm
Figure 11
The values represent the means of 10 determinations + standard
deviation.
Wet weight of male and female S.marqrebowiei over a period from
42 - 84 days post infection.
Figure 12
Protein content of male and female S.marqrebouiei over a period
from 42 - 84 days post infection.
The values represent the means of 10 determinations + standard
deviation
pg p
rote
in /
wor
m
mg
wet
wei
ght
/ w
orm
Fig. 1 1
Fig. 12
Table 5
Comparison of Lowry and Bradford protein assay techniques.
♦
4
m
m-
STANDARD LOWRY BRADFORD
20 1 9 + 2 (5) 13 + 0*3 (5)30 2 6 + 2 (5) 21 + 0.4 (5)50 44 + 1 (5) 39 + 1 (5)70 ' 67 + 2 (5) 58 ± 5 (5)
100 96 + C M (5) 90 ± 1 (5)
Values = pg protein + standard error, with the number of
determinations in parentheses.
Fiaure 13
Protein : liiet weight ratios in male and female S.mansoni over a period from 42 - 84 days post infection.
The values represent the means of 10 determinations + standard
deviation.
Figure 14
Protein : Wet weight ratios in male and female S.margrebowiei
over a period from 42 - 84 days post infection.
The values represent the means of 10 determinations + standarddeviation.
Fig.13
Fig. 14
Figure 15
Total lipid content of male and female S.mansoni over a period
from 42 - 84 days post infection.
The values represent the means of 10 determinations + standarddeviation
Fig. 15
1600 " i
Days post infection
TOTAL LIPIDS3,7.
Levels of total lipids showed no statistical changes between 42 - 84 DPI in male S.mansoni (Fig. 15), whereas the levels in
female S.mansoni showed an overall decline over the same period
(Fig. 15). Total lipid content in both sexes showed no statistical
difference until 84 DPI when the level in males was 82$ higher than in females.
3.8. ENZYME ANALYSIS
3.8.1. S.MANSONI AND S.MARGREBOWIEI
3.8.1.1. Glycolysis
The activities of glycolytic enzymes of S.mansoni and S.marqre-
bowiei were higher than enzymes of the tricarboxylic acid (TCA)
cycle and hexosemonophosphate (HMP) shunt (Tables 6, 7, 8, 9 and 10).
In addition the glycolytic enzyme activities of male schistosomes
were higher than those of the females (Fig. 6). The phosphoenol-
pyruvate carboxykinase (PEPCK): pyruvate kinase (PK) activity ratios of male and female S.mansoni and S.margrebowiei were 0*1,0»3, 0.01 and 0»002, respectively. The activities of malic enzyme,
fumarase and nucleoside diphosphokinase of both species were either
low or not detectable and lactate dehydrogenase activities were relatively high (Tables 6 and 7). This indicates that lactate
formation is a more important route of energy synthesis than CO2
fixation and the TCA cycle in these parasites. Malate dehydrogenase (OAA - MAL) in males and females of bothnspecies was very active
in comparison with other enzymes (Tables 6 and 7). The alternative terminal glycolytic enzymes i.e. octopine dehydrogenase and alcohol
dehydrogenase were not detectable in either species.
3.8.1.2. TCA cycle
TCA enzymes were more easily detectable in S.marqrebowiei than
in S.mansoni, although isocitrate dehydrogenase was not measurable
in either species (Tables .8 and 9). Citrate synthase-activity in female S.mansoni increased significantly (P<0*05) in the pre
sence of Triton x - 100 (10 pl/ml)(Table 8 ). Pyruvate dehydrogenase activity was not detectable in homogenates of S.mansoni even in the presence of Triton x - 100 (10 pl/ml) and 5 mM MgC^ at protein levels of up to 50 pg/cuvette.
3.8.1.3. HMP shunt
Glucose -6- phosphate dehydrogenase and 6- phosphogluconate
dehydrogenase activities were detectable in both species of
schistosome (Table 10). In addition, transaldolase activity was measurable in S.mansoni homogenates.
3.8.2. ENZYME ACTIVITIES IN OTHER SPECIES
Glycolytic, TCA and HMP enzyme activities in males and females of S.bovis, S.haematobium (S.African strain), S.intercalatum,
Table 6
Glycolytic and associated enzyme activities in male and femaleS.mansoni
ENZYME MALES FEMALES
Phosphorylase a + b 64 ± 0 (3) 92 + 13 (3)Hexokinase 130 + 19 (3) 89 + 11 (3)Phosphoglucomutase 1113 + 59 (3) 388 ± 13 (4)Glucosephosphate isomerase 49 + 3 (4) 12 + 2 (4)Phosphofructokinase 10 + 1 (4) 40 ± 10 (4)Aldolase 466 + 23 (4) 139 + 14 (4)Glycerol-3-phosphatedehydrogenase 442 ± 48 (3) 361- + 39 (3)Triosephosphate isomerase 5811 ± 114 (4) • 2063 + 458 (4)Glyceraldehyde-3-phosphatedehydrogenase 183 ± 15 (4) 56 ± 2 (4)Phosphoglycerate kinase 459 + 42 (4) 108 ± 6 (4)Phosphoglycerate mutase 3645 + 216 (4) 2475 ± 50 (4)Enolase 2162 + 291 (3) 1775 + 268 (3)Pyruvate kinase 370 ± 4 (7) 182 + 34 (3)L-lactate dehydrogenase 9314 + 1291 (6) 610 ± 100 (5)
Phosphoenolpyruvatecarboxykinase 37 + 6 (10) 58 ± 4 (10)Alcohol dehydrogenase n.d. n.d.Octopine dehydrogenase n.d. n.d.Malate dehydrogenase (OAA-MAL) inCDCD<r + 238 (5) 957 ± 101 (5)Malic enzyme (MAL- PYR) n.d. n.d.Malic enzyme (PYR-MAL) n.d. n.d.Nucleoside -5'- diphosphokinase 34 ± 2 (4) 32 ± 1 (4)Glutamate-oxaloacetatetransaminase 17 ± 3 (3) 32 ± 5 (3)n.d. = activity not detectable
Results are expressed as nmoles product formed/min/mg protein + standard error. Figures in parenthesis represent the number ofdeterminations.
Table 7
Glycolytic and associated enzyme activities in male and female
5.margreboudei.
4ENZYME MALES FEMALES
*
*
Phoshorylase a + bHexokinasePhosphoglucomutaseGlucosephosphatsPhosphofructokinaseAldolaseGlycerol-3-phosphate dehydrogenase Triosephosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase EnolasePyruvate kinase L-lactate dehydrogenasePhosphoenolpyruvate carboxykinase Alcohol dehydrogenase Octopine dehydrogenase Malate dehydrogenase (OAA-MAL) Malic enzyme (MAL- PYR)Malic enzyme (PYR-MAL)
11146
±±
0-01 3) 15 (3)
2414
++
0-01 (3) 67 (3)
1086 ± 100 (3) 776 ± 97 (3)4546 ± 366 (3) 2319 + 118 (3)1464 ± 35 (3) 561 + 44 (3)
888 ± 79 (3) 448 + 18 (3)325 + 10 (3) 178 + 9 (3)
3698 ± 876 (3) 2789 + 641 (3)1292 + 56 (3) 959 + A0 (3)213 + 1 (3) 22 + 1 (3)
1449 ± 333 (3) 1264 + 269 (3)256 ± 30 286 ± 24 (3)155 + 10 (4) 577 + 151 (3)2056 + 318 (it) 2004 ± 401 (4)
2 + 1 (4) 1 + 0*2 (4)n,.d. n.,d.n,,d. n.,d.
712 ± 165 (5) 628 + 106 (5)n,,d. n,.d.n,,d. n..d.
n.d. = activity not detectable
Results are expressed as nmoles of product formed/min/mg protein + standard error. Figures in parentheses represent the number ofdeterminations
#
Table 8
♦
Tricarboxylic acid cycle enzyme activities in male and female
S.mansoni.
#
ENZYME MALES FEMALES
Pyruvate dehydrogenase n.d. n.d.(6)Citrate synthase 2 + 1 (6) 2 ± 1
Citrate synthase + Triton x - 100 - 5 + 0*1 (3)Aconitase ^ 2 ± 1 (4) 11 ± 2 (4)Isocitrate dehydrogenase (Mi?,NAD)
" " (Mq ,NADP)n.d. n.d.n.d. n.d.
,f " (Mn,NAD) n.d. n.d." " (Mn,NADP) n.d. n.d.
2-0xoglutarate dehydrogenase n.d. n.d.Succinate dehydrogenase (SUCC - FUM) n.d. n.d.Succinate dehydrogenase (FUM -SUCC) n.d. n.d.Fumarase n.d. n.d.Malate dehydrogenase (MAL - OAA) 31 + 4 (4) 29 + 9 (4)
n.d. = activity not detectable
= assay not performed
Results are expressed as nmoles of product formed/min/mg protein
+ standard error. Figures in parentheses represent the number of
determinations
Table 9
Tricarboxylic acid cycle enzyme activities in male and female
S.marqrebouiei.
*
m
♦
ENZYME
Citrate synthase AconitaseIsocitrate dehydrogenase (Mcf,NAD)
" n (Mq,NADP)" " (MiT,NAD)
2-Qxoglutarate dehydrogenase Succinate dehydrogenase (SUCC - FUN) Succinate dehydrogenase (FUM-SUCC) FumaraseMalate dehydrogenase (MAL- OAA)
MALES FEMALES
10 ± 1 (5 ) 25 + 5 (4 )0.2 + 0 *03 (3) 0-2 ± 0-1 (3 )
n,,d. n,,d.n,,d. n,,d.n,.d. n,,d.
12 + 11 (3) 17 + 12 (3 )a + 1 (3 ) 10 + 1 (3 )
0*2 ± o.1 (3 ) 0-3 0-1 (3 )1 + 0 «2 (3) 2 + 0*2 (3 )
13 ± 3 (5 ) 17 + 5 (4 )
n.d. = activity not detectable
Results expressed as nmoles product formed/min/mg protein +
standard error. Figures in parentheses represent the number of
determinations.
*
m
Table 10
Hexosemonophosphate pathway enzyme activities in male and female
S.mansoni and S.margrebowiei.
S.MANSQNI S .MARGREBOUilEI
ENZYF1E MALES FEMALES MALES FEMALES
Glucose-6-phosphate dehydrogenase 82 + 15 (A) 3 5 + 2 (4) 166 + 7 (3) 126 + 1 (3)6-Phosphogluconate dehydrogenase 83 ± 14 (4) 9 + 2 (3) 3 7 + 2 (3) 37 + 1 (3)
Transaldolase 2 2 + 3 (4) 22 + 1 (A) - -
- = assay not performed.
Results are expressed as nmoles product formed/min/mg protein + standard error. Figures in
parentheses represent the number of determinations.
Table 11
Enzyme activities in male S.bovis, 5.haematobium, S.intercalatum,
S.japonicum and S.leiperi.
Results are expressed as nmoles product formed/min/mg protein +
standard error. Figures in parentheses represent the number of
determinations
ENZYME S.BOVIS
Glucosephosphate isomerase Triosephosphate isomerase Glycerol-3-phosphate dehydrogenase Pyruvate kinase L-lactate dehydrogenase
5 + 1 (7) 785 + 110 (7) 335 + 10 (7)
Phosphoenolpyru\/ate carboxykinase Malate dehydrogenase (OAA-MAL)
" n (ma l-o a a)Citrate synthaseIsocitrate dehydrogenase (Mg,NAD)
" " (Mg,NADP) " " (Mn,NAD)" " (Mn,NADP)
2-0xoglutarate dehydrogenase Fumarase
3 3 + 4 (7) 4 7 + 5 (6) 16 + 1 (5)
n.d. n.d. n.d. n.d.
Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase
147 + 18 (7) 3 7 + 6 (5)
n.d. = activity not detectablei
= assay not performed.
u Species originated from Durban, South Africa.
f 0-ip *
S. HAEMATOBIUM# S. INTERCALATUM S. J APONICUM
- - 24 + 1 (3)- - 60 + 1 (3)- 82 + 20 (6) -
• 11 (6) 5419 + 447 (7) 486 + B7 (3); 87 (0) 4925 + 566 (7) 797 + 25 (3)
- - 20 + 3 (3)- 2727 + 194 (7) 818 + 41 (3)- 44 + 5 (5) 26 + 5 (3)- 64 + 6 (5) n,.d.
n.d. 3 + 1 (3) -
n.d. n,,d. -
n.d. n,,d. -
n.d. n,.d. -
- - 5 + 0*3 (3)- - n,,d.- 62 + 2 (3) 59 + 4 (3)- 39 + 1 (3) 0 + 2 (3)
S.LEIPERI
n.d.1355 ± 480 (3)2524 + 879 (3)2284 + 977 (3)
1 8 + 4 (3) 1 7 + 3 (4)
n.d. n.d. n.d. n.d.
44 + 16 (4) 11 + 1 (4)
Table 12
Enzyme activities in female S.bouis, 5.haematobium, S.intercalatum,
5,.japonicum and S.leiperi.
Results are expressed as nmoles product formed/min/mg protein +
standard error. Figures in parentheses represent the number ofdeterminations
ENZYME S.BOVIS
Glucosephosphate isomerase Triosephosphate isomerase Glycerol-3-phosphate dehydrogenase Pyruvate kinase L-lactate dehydrogenase Phosphoenolpyruvate carboxykinase Malate dehydrogenase (OAA - MAL) Malate dehydrogenase (MAL - OAA) Citrate synthaseIsocitrate dehydrogenase (McnNAD)
" " (Mg,NADP)" " ( m , M Q )
" " (Mn,NADP)2-Qxoglutarate dehydrogenase FumaraseGlucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase
3 + 0-2 (7)403 + 96 (6)258 + 5 (7)23 + 2 (7)65 + 13 (4)35 + 5 (4)
n«.d.n,,d.n,,d.n.,d.
113 + 24 (5)90 ± 15 (5)
n.d. = activity not detectable.
= assay not performed
* Species originated from Durban, S.Africa
* ♦
S.HAEMATOBIUM* S.INTERCALATUM S.JAPONICUM S.LEIPERI
- 191 + 42 (3)416 + 82 (3) 2405 + 435 (3)38 ± 12 (3) 2775 + 255 (3)
_ 1278 + 75 (3)- 47 ± 2 (3)- 31 + 3 (3)- n.d.- n.d.- n.d.— n.d.—
74 + 3 (3)- 33 + 3 (3)
19 + 1 (3) _
52 + 2 (3) -
- n.d.480 + 80 (3) 1308 + 460 (3)180 + 17 (3) 1796 + 597 (3)25 + 3 (3) -
739 + 61 (3) 1737 + 562 (3)26 + 5 (3) 29 + 3 (3)
n,,d. 24 + 17 (3)- n,,d.- n,.d.- n,.d.- n.,d.
7 + 1 (3) -
n..d. -
54 hh 3 (3) 46 + 3 (3)12■ it 1 (3) 11 + 2 (3)
5.japonicum and S.leiperi showed similar relationships to those
observed for S.mansoni and S.mararebowiei (Tables 11 and 12).
The malate dehydrogenase activities (OAA - HAL) in all species except S.bovis were high in comparison u/ith other enzymes (Tables
11 and 12). The lactate dehydrogenase activities of male and
female S.bovis and 5.haematobium were lower than the activities
of the same enzyme from other species. Isocitrate dehydrogenase
activity was only detected in male S.intercalatum (Table 11) and
glucose -6- phosphate dehydrogenase measurable in all species
investigated.
3.9. METABOLITES
3.9.1. ANALYSIS OF METABOLITES OF MOUSE LIVER
The comparative levels of selected metabolites assayed by spectrophotometry or fluorimetry showed no significant differences
(Table 13). The levels of fructose -1, 6- bisphosphate (FBP) and
pyruvate were significantly lower (P<0*02) than those published
by Williamson and Brosnan (1974) but the levels of phosphoenol-
pyruvate (PEP) and 2- phosphoglycerate (2 - PG) were found to be significantly higher (P«=0*01j.
3.9.2. ANALYSIS OF STANDARD METABOLITE SOLUTIONS
Table 14 shows the results of spectrophotometric and fluori- metric assay methods for measuring known concentrations of selected
Table 13
Comparison of metabolite levels in mouse liver measured by
spectrophotometry and fluorimetry.
METABOLITE SPECTROPHOTOMETRY FLUORIMETRYWILLIAMSON # & BROSNAN
Fructose - 1, 6-bisphosphate 7 + 3 (3) 5 ± 1 (3) 24
Pyruvate 152 + 5 (3) 145 + 4 (3) 170
Phosphoenolpyruvate 273 + 18 (3) 224 + 12 (3) 110
3-Phosphoglycerate 263 t 8 (3) 270 + 3 (3) 220
Values = nmoles/g wet weight + standard error, with the number of determinations in parentheses.
* = Data taken from Williamson & Brosnan (1974).
Table 14
Comparison of standard solutions of metabolites measured by
spectrophotometry and fluorimetry.
METABOLITE STANDARD SPECTROPHOTOMETRY FLUORIMETRY
Oxaloacetate 140 145 + 15 (3) 137 ± 4 (3)Pyruvate 120 123 ± 1 (3) 119 + 4 (3)2-Phosphoglycerate 100 97 + 3 (3) 103 + 6 (3)
Values = nM + standard error, uith the number of determinations in parentheses.
metabolites. No significant differences were found between the
data obtained using both techniques.
3.9.3. ANALYSIS OF PARASITE METABOLITE LEVELS
3.9.3.1. S.mansoni
Table 15 shows the metabolite levels in male and female
S.mansoni using a combination of fluorimetric and spectrophoto-
metric assays. All the glycolytic intermediates investigated
were detected and L-lactate was by far the most abundant metabolite
detectable in both sexes of schistosome. Of the TCA cycle intermediates only oxaloacetate (OAA) was detectable. OAA levels in
male worms were significantly higher (P«=0*02) than in females. Similarly, ATP levels were far higher in males than in females
(P«0»01) but AMP levels were higher in the females (P«=0*Q2).The adenylate charge and ATP:AMP ratios were both higher in male
parasites than in females (Table 17).
3.9.3.2. S.marqrebowiei
All the glycolytic intermediates investigated in S.marqrebowiei were detected except dihydroxyacetone phosphate (DHAP) in the
females (Table 16). L-lactate levels were relatively high and
similar in both sexes. OAA was the only TCA cycle metabolite
measurable and levels were significantly higher (P«=0#01) in the males. Similarly DHAP and 3- phosphoglycerate (3-PG) levels were also higher in male parasites. ATP, ADP and AMP levels were not
Table 15
Metabolite levels in male and female S.mansoni.
METABOLITE MALES
Fructose -1, 6-bisphosphate 23 ± 7 (11)Dihydroxyacetone phosphate 9 + 1 0 )Glyceraldehyde-3-phosphate 5 + 1 (8)3-Phosphoglycerate 33 + 9 (5)2-Phosphoglycerate 54 ± 12 (5)Phosphoenolpyruvate 26 + 7 (10)Pyruvate 31 + 9 (12)Lactate# 5553 + 680 (6)
DL-Isocitrate n.d.
2-0xoglutarate n.d.
Oxaloacetate 1894 + 385 (5)
ATP# 2184 + 119 (3)
ADP# 529 + 420 (4)AMP# 14 ± 1 (3)
Values = nmoles/g wet weight + standard error, with determinations in parentheses.
# = metabolites assayed by spectrophotometry.
50 + 18 (4)
88 + 33 (4)
3 + 3 (5)
2 0 + 3 (3)32 + 10 (4)
1 8 + 2 (4)
33 + 12 (4) 4247 + 1544 (6)
n.d.
n.d.
4 5 + 7 (3)
631 + 23 (3)
426 ± 16 (3) 179 + 17 (3)
the number of
FEMALES
n.d = not detectable.
Table 16
Metabolite levels in male and female 5.margrebowiei.
METABOLITE MALES FEMALES
Fructose -1, 6-bisphosphate 14 + 4 (5) 28 + 11 (3)Dihydroxyacetone phosphate 72 + 26 (3) n,>d.Glyceraldehyde-3-phosphate 7 ± 3 (3) 2 + 0*2 (3)3-Phosphoglycerate 59 + 9 (3) 4 + 1 (4)2-Phosphoenolpyruvate 32 + 12 (6) 8 + 3 (3)
Phosphoenolpyruvate 11 + 3 (4) 6 ± 1 (3)Pyruvate 10 + 6 (5) 12 + 5 (3)Lactate # 480 + 280 (6) 876 + 472 (6)DL-Isocitrate n.-d. n,.d.2-0xoglutarate n,.d. n.>d.Oxaloacetate 677 + 185 (5) 6 + 2 (4)
Values = nmoles/g wet weight + standard error, with the number of
determinations in parentheses,
# = metabolite assayed by spectrophotometry.n.d = not detectable
Table 17
Adenylate charge and ATP : AMP ratios in S.mansoni
MALES FEMALES
Adenylate charge 0*89 0*70
ATP : AMP 156 3*53
Adenylate charge = [ATP] + 2 [AD§[AMP + [ADP] + [A TPJ
measured in S.marqrebowiei due to lack of material.
A number of interspecific differences were revealed in this
study. L-lactate and QAA levels were significantly higher ((P**0*001) and (P<0*02) respectively) in male S.mansoni than in male
5.marqrebowiei. However, the latter contained higher levels of DHAP (P<0*05). Female S.mansoni contained higher levels of OAA
(P-s=0«01), DHAP (P«=cO*OT) and PEP (P<CU01) than female S.marqreboujiei.
3.9.4. Hass action ratios
The available data was used to calculate the mass action ratios
(MAR), of a few glycolytic enzymes in S.mansoni and S.marqrebowiei.
The MAR is the ratio of concentrations of products:reactants in
a given reaction (Price & Stevens, 1982). The MAR: equilibrium
constant ratios for both male and female S.mansoni PKs was 1 x 10"^ and so according to Rolleston (1972) the enzymes are catalysing
non-equilibrium reactions in vivo. The other enzymes i.e. aldolase, triosephosphate isomerase, 2-phosphoglycerate mutase and enolase
were, as expected, regulating equilibrium reactions.
3.1Q. INCUBATIONS
3.10.1. S.MANSONI
3.10.1.1. Glucose uptake and lactate productionof L-lactate produced by female and paired S.mansoni
was not significantly different from the amount.of glucose con
sumed from the medium and endogenous sources (Fig. 16). The
glucose consumed:lactate^produced (G:L) ratios were 1:1 *0 and
1:0.9 respectively. In single males, lactate production was
significantly greater than glucose consumption (P«= 0*001) and
the G:L ratio was 1:2*2 (Fig. 16). The comparative levels of
glucose consumption over the 6h incubation period were in the order; pairs, single females (P<0*05) single males (P< 0*002)
and the comparative levels of lactate production were in the order pairs, single females (P«=0*02) single males (P<0*001) (Fig.16). The initial incubation pH level of 7*5 fell to an average of 7*2
+ 0*1(9 determinations) after the experiment.
3.10.1.2. Metabolite levelsAlanine, acetate, citrate, ethanol, glycerol, D-lactate,
oxaloacetate, pyruvate, propionate and succinate were not detectable in incubation samples. The only endogenous metabolites
detectable were glucose, glycogen, glycerol and L-lactate (Figs.
17 and 18). Endogenous glucose and glycogen levels in female S.mansoni showed no significant change during the incubation but
levels in males showed a marked reduction of 56$ (P<0*05) and 55$ (P<0*001) for glucose and glycogen respectively (Fig. 17).
Glycerol and L-lactate levels in female S.mansoni did not change
during the incubation (Fig. 18). A similar situation was found
with glycerol levels in male S.mansoni but the quantities of
endogenous L-lactate declined by 82$ (P-=0*001).
143.10.1.3. COq productionPaired and single S.mansoni liberated ^ C 02 from D - U -
Figure 16
Glucose consumption from the medium and L-lactate production
by S.mansoni during 6h incubation. The values represent the
means of 9 determinations + standard error. The data represents
the values obtained after correcting for changes in endogenous
metabolite levels and controls.
Figure 17
Endogenous glucose and glycogen levels in S.mansoni before and
after 6h incubation. The values represent the means of 9
determinations + standard error.
Fig. 16
Glucose consumption | | L-lactate production urn
FigOZ
Before incubation! 1 After incubation\//A
Glucose ^ Glycogen
Figure 18
Endogenous L-lactate and glycerol levels in S.mansoni before
and after 6h incubation.The values represent the means of 9 determinations + standarderror
nmol
es /
mg
prot
ein
Fig. 18
♦
%
Before incubationj 1 After incubation (ZZZ
1 2 0 -
80-
40-
$
L-lactate Glycerol
♦
Figure 19
CO2 production by S.mansoni and mouse blood during 6h incubation.
The values represent the means of 6 determinations, accounting for
controls, + standard error. The data is expressed in terms of the
amount of glucose that would be consumed if the TCA cycle alone
is operating.
14
Figure 20
14D - U - C glucose incorporation into the tissues of S.mansoni during 6h incubation.
The values represent the means of 6 determinations, accounting
for controls, + standard error.
<#* 4ft-
nmoles glucose incorporated / mg protein
Fig.20
Mouse blood
w 0 04Ji>
6r§!d
glucose (Fig. 19). Single male parasites produced significantly / > 1Amore (P<001) CC^ than single females or paired worms. Paired
14worms and single females showed no difference in levels of CC^
production (Fig. 19). House whole blood controls also produced14 / \low levels of CC^ during incubation (Fig. 19).
3.10.1.4. Glucose incorporation14Single male and female S.mansoni incorporated D - U - C
glucose into body tissues with the males showing higher levels of incorporation (P<0*002) than the females (Fig. 20).
3.10.2. S.MARGREBOUJIEI
3.10.2.1. Glucose uptake and lactate production
The quantity of L-lactate produced by single and paired worms
was significantly different (P<0«02) from the amount of glucose
consumed. Lactate production in single males was greater than
glucose consumption with the G:L ratio being 1:1*7 (Fig. 21).
In contrast, single females and paired worms consumed more glucose
than they produced lactate and the G:L ratio was 1:0*7 for both
groups (Fig. 21). The comparative order of glucose consumption during incubation was as follows :- single females and paired
worms, single males (P<0*01) and the order for lactate production was:- single males, paired worms (P<0*05). There was no statistical difference in lactate production between single males and females or single females and paired worms. The initial pH level
Figure 21
Glucose consumption from the medium and L-lactate production
by S.marqrebouiiei during 6h incubation.The values represent the means of 3 determinations + standard
error. The data represents the values obtained after correcting
for changes in endogenous metabolite levels and controls.
Figure 22
Endogenous glucose and glycogen levels in S.marqrebouiiei before
and after 6h incubation.The values represent the means of 3 determinations + standard
error.Vfr GnS'yc&cyZ-'A, vJc\Wv\\c„
pmol
es /
mg
prot
ein
Hm
oles
/ m
g pr
otei
nFig.21Glucoseconsumption
L*lactateproduction EZ2
Fig.22
Before incubationl | After incubationT 7 7 7 \,
<? ? Cf $
Glucose * Glycogen
Figure 23
Endogenous L-lactate levels in S.margrebowiei before and after
6h incubation.
The values represent the means of 3 determinations + standard
error.
Figure 24
14 .CO2 production by S.marqrebowiai during 6h incubation.The values represent the' means of 3 determinations minus controls,
+ standard error. The data is expressed in terms of the amountof glucose that would be consumed if the TCA cycle alone is
operating.
(+
nmoles glucose consumed / mg protein
Fig. 24
♦
nmoles / mg protein
CD<Do-ICD=3OcCT 0)o'3
Fig.23
102
*
*
w
of the medium (7*45) fell to an average of 7*1 + 0-4 (3 determin
ations) after incubation.
3.10.2.2. Metabolite levelsAnalysis of the incubation medium failed to detect any
metabolites except glucose and L-lactate. Moreover, glucose,
glycogen and L-lactate were the only endogenous metabolites detectable (Figs. 22 and 23). Glucose levels in single males and
females showed no significant changes during incubation. A
similar situation occurred with glycogen levels in single male
worms but the females showed a significant increase (Fig. 22).
Endogenous lactate levels in male worms remained constant but the
females showed a significant decrease (P<0*01) during incubation
(Fig. 23).
143.10.2.3. Co-production14 14Each experimental group produced CO^ from the D -U - C
glucose in the medium and the comparative levels of production were single females, single males and paired worms (P<0*01) (Fig.24).
3.10.3. S.HAEMATOBIUM
3.10.3.1. Glucose uptake and lactate production
In single male and female worms, glucose consumption was
significantly higher ((P<-0*02) and (P<0*001) respectively) than L-lactate production and the respective G:L ratios-were 1:0*4 and
1:0*5 (Fig. 25). Glucose depletion by female worms was higher
than for males (P«=0*01) and the same occurred with lactate production (P<0*05) (Fig. 25). The initial pH of the medium (7*6)
fell to an average of 7 + 0*2 (2 determinations) after incubation.
3.10.3.2. Metabolite levels
The end product metabolites assayed in the medium in addition
to L-lactate were succinate, pyruvate and acetate. The latter
was detectable in low but similar levels in male and female worms
(Fig. 25). Glucose and glycogen were the only endogenous meta
bolites assayed. Glucose levels in male and female parasites
increased significantly during incubation (P<0*05) (Fig. 26), whereas no changes were observed in the glycogen levels of either
sex (Fig. 26).
3.10.4. S.douthitti
3.10.4.1. Glucose uptake and lactate production
Female parasites were not available for study as the material
was obtained from a unisexual infection. L-lactate production in single male S.douthitti was significantly higher (P<0*02) than glucose consumption and the G:L ratio was 1:2*4 (Fig. 27).
3.10.4.2. Metabolite levelsEndogenous glucose levels remained constant during incubation
but glycogen was completely depleted (Fig. 28). Tissue levels
Figure 25
Glucose consumption from the medium, L- lactate and acetate
production by S.haematobium (Morroccan strain) during 6h
incubation.
The values represent the means of 3 determinations + standard
error. The data represents the values obtained after correcting for changes in endogenous metabolite levels and controls.
Figure 2G
Endogenous glucose and glycogen levels in S.haematobium (Morroccan)
before and after 6h incubation.
The values represent the means of 3 determinations + standard
error.
£iSi25
(f ? cf $
Fig.26
Before incubationL___ l After incubation E Z 2
d 1 £ d 1 2
Glucose * Glycogen
Figure 27
1AGlucose consumption from the medium,L-lactate and CO^
production by male S.douthitti during 6h incubation.
The values represent the means of 3 determinations + standard
error. The data represents the values obtained after correcting14for changes in endogenous metabolite levels and controls. CC^
production is expressed in terms of the amount of glucose that
would be consumed if the TCA cycle alone operating.
Figure 28
Endogenous glucose, glycogen and L-lactate levels in male
S.douthitti before and after 6h incubation.
The values represent the means of 3 determinations + standard
error.
pmol
es /
mg
prot
ein
Fig.27
production
Fig.28
Before incubation f I After incubation V / A
Glucose Glycogen L-1 act ate
of L-lactate did not change significantly during incubation (Fig, 28)
D-lactate, alanine, acetate, glycerol, pyruvate, propionate and
succinate were undetectable in tissue or media samples.
143.10.4.3. COq productionMale S.douthitti produced low levels of ^CO^ from D- U -
glucose during incubation (Fig. 27).
3.10.5. Interspecific comparisons5.haematobium males and females consumed the most glucose
during 6h incubation followed by S.marqrebowieir S.mansoni and
S.douthitti. Exogenous glucose appeared to be used in preference
to endogenous sources as there were no large depletions in glycogen
except with S.douthitti. In each case, female schistosomes pro-14duced at least as much lactate as their male counterparts. CO2
was produced in each species investigated hence suggesting thatsome oxidative pathway is functioning but the levels indicate
that the pathway is operating at a low rate. The mouse blood14controls also produced CC^ but not enough to seriously interfere
with the experiment. The levels of mouse blood contamination
will be even lower in the actual experimental tubes as the
number of worms used would contain smaller volumes of gut contents.
Radiolabelled glucose was incorporated into worm tissue during incubation thus suggesting that glucose is not solely
used for catabolic processes. The pH levels of the media also
remained within those described as being optimal by Bueding (1950).
Overall it was found that male parasites were using almost all
their available glucose for glycolysis but the females were channelling a large proportion (approximately 50%) of their
glucose into alternative pathways.
3.11. KINETIC STUDY OF PYRUVATE KINASEIt was decided to draw in lines of best fit by 11 eye" for
all pyruvate kinase (PK) saturation curves and to rely on logarithmic or inverse transformations to derive Hill Co-efficients
(HCo) and apparent Km (K1) and Umax (^m) values.
3.11.1. S.MANSQNIThe saturation curves for male and female enzymes, were
sigmoidal and rectangular hyperbolic respectively (Fig. 29); the data transformed to Hill plots is shown in Eig. 30. The K! and
HCo values were determined as set out in Fig. 30 and are shown
in Table 18. The respective HCo values for male and female PK were 1*20 and 0»96 thus according to Price & Stevens (1982) and
Lehninger (1977) the enzymes exhibited positive and negative
co-operativity respectively. Yet, as the HCo of the female enzyme was nearly 1 (i.e. denoting normal Michaelis/Menten
kinetics) the data has been transformed to a Woolf (Hanes) plot
(Price & Stevens, 1982) (Fig. 31). The K! value for male PK was lower than that of the females (Table 18), indicating that the
male enzyme has a higher affinity for phosphoenolpyruvate (PEP). The Vmax for the male enzyme was also higher than that for the
Figure 29
The effect of PEP concentration on the activity of pyruvate kinases from male and female S.mansoni.The values represent the means of 6 determinations + standard
error
*
♦
*
♦
T1
14
Figure 3D
Hill plot of the effect of PEP concentration on the activity
of pyruvate kinases from male and female S.mansoni.
Slope=n=Hill coefficient
Intercept Y =-log. K* = -nlog,o [PEd‘° 50
[PEf = concentration at Vmax/2 50
K* = Apparent Km
110
#
Figure 31
Woolf (Hanes) plot of the effect of PEP concentration on the
activity of pyruvate kinase from female S.mansoni.I
*
#
*
0
[PEP] mM
Intercept X = —Km
Intercept Y = Km/Vmax
%
female (Table 18).
3.11.1.1. ATP inhibition
In male S.mansoni 1nM ATP had no effect on PK activity but
between 1nM - 1|jffl there was a 23? inhibition and between 1pM - 1mM
there was a further 23? inhibition (P<0»001) (Table 19). Simi
larly, female PK is not inhibited by 1nM ATP but there was a
significant decline (P<0*002) of 19? in activity at 1pM. No
further inhibition occurred at 1mM ATP (Table 19), therefore, the
female enzyme is less susceptible to inhibition than male PK over
the entire ATP concentration range but equally as sensitive
between 1nM - 1pM ATP.
3.11.1.2. FBP activation
Male and female PKs both showed increased activity in the
presence of FBP (Table 19). Male PK showed a significant overall
increase of 59? from 1 pM — 25|jM FBP (P<0«001). There was a 10?
decrease in activity from 25jjM - 125pM FBP, suggesting that 25jjM
FBP is the concentration at which maximal activation occurs.
Female PK showed a 54? increase in activity from 0 — 1 jjM FBP but
from 1pM - 25jjM there was no further significant change in activity.
From 25jjM - 50|jM FBP there was a further increase of 35? but there
was no significant change in activity from 50|jM - 125|jM (Table 19).
Thus it appears that an FBP concentration of 50|jM is necessary
for maximal activation of the female enzyme. In comparison with
the male enzyme, female PK required twice the concentration of FBP
to achieve maximal and similar levels of activity. The respec
tive levels of FBP which cause maximal activation are similar to the endogenous levels in the parasites (section 2.9.1.).
3.11.2. S.MARGREBQWIEIThe saturation curves of PKs from males and females were
both sigmoidal (Fig. 32) and the Hill plots are shown in Fig.33.
The male enzyme was approximately twice as active as the female at PEP concentrations from 2mM - 5mM. The K* for male PK was
lower than for female PK and the male enzyme also had a higher
HCo (Table 18). Hence the male enzyme appears to be more positively co-operative and has a higher affinity for PEP than
the female enzyme.
3.11.2.1. ATP inhibition
Male PK showed no significant alteration in activity at
1nM ATP (Table 20). Between 1nM - 1pM there was a significant (P<0*02) 27% decrease in activity and from 1pM - 1mM ATP there
was no further change. Female PK showed no significant change in activity from 0 - 1pM ATP but from 1pM - 1mM there was a
significant (P<0-01) decrease of 41% (Table 20). Therefore,
male PK appears to be inhibited by lower levels of ATP.
3.11.2.2. FBP activation
PK activity of males and females increased in the presence
of FBP (Table 20). Male PK activity was increased by a factor
Figure 32
The effect of PEP concentration on the activity of pyruvate
kinases from male and female S. margrebouiei.
The values represent the means of 4 determinations + standarderror
5000
•5 4000-
bo
^ 3000-cE
(/>0)oEc
2000-
< 1000 -
*
?T
— j------------------------ 1-------------------------- 1
3 4 5
[PEFj mM
•qj|> a #
Figure 33
Hill plot of the effect of PEP concentration on the activity
of pyruvate kinases from male and female S.marqrebouiei.
of 28 from 1nM - 1mM FBP and the activity of the female enzyme
showed a 23 fold enhancement over the same range. The male enzyme
was more significantly activated (P<0*001) over the range InM -
1|#1 than the female enzyme, but in the range - ImM female
PK was more significantly activated (P<=001) than the male enzyme.
3.11.3. 5.JAPDNICUMThe saturation curves of PKs from males and females were
both sigmoidal (Fig. 34) and the Hill plots are shown in Fig. 35.
The HCo and K ! for the male enzyme were higher than those for the
female, thus indicating that the former has a comparatively lower
affinity for PEP and appears to: be more positively co-operative
(Table 18).
3.11.4. S.BQVI5
It was not possible to determine whether the saturation
curve for the male enzyme was a sigmoid or a rectangular hyper
bola (Fig. 36). This was because the lowest recorded activities did not allow the determination of the shape of the lower portion
of the curve. Therefore the available data was transformed to
Hill and Woolf (Hanes) plots (Figs. 37 & 38 respectively) and the derived K* and U’m values shown in Table 18. The K ’ from the Hill plot appeared to be rather high and so the alternative
data was regarded as being more appropriate.
Figure 34
The effect of PEP concentration on the activity of pyruvate
kinases from male and female 5.japonicum.
The values represent the means of 5 determinations + standard
error.
c0)4—*ov_CLboEc
CO0)oEc
o<
20 0 0 -
1600
1200-
800-
400
4
$ ,
--------------------- ,--------------------------1--------------------------1
3 4 5
[pEP]mM
4 ♦ *
Figure 35
Hill plot of the effect of PEP concentration on the activity
of pyruvate kinases from male and female 5. japonicum.
log [PEI mM
71
1 1 8
Figure 36
The effect of PEP concentration on the activity of pyruvate*kinase from male S.bovis.
The values represent the means of 5 determinations + standard
error.*
m
*
[PEP] m M
F ig u re 374
Hill plot of the effect of PEP concentration on the activity
of pyruvate kinase from male S.bovis.
4
Figure 38
UJoolf (Hanes) plot of the effect of PEP concentration on the activity of pyruvate kinase from male S.bovis.
•#
4
F ig .3 7
Fig. 38
[P E P ] mM
Table 18
Apparent Km (K!) and Vmax (\y*m) values for the pyruvate kinases
from S.mansoni, 5.marqreboiuiei, S./japonicum, S.bovis and mouse
blood.
S.MANSONI S.MARGREBOUIEI S.JAPONICUM S.BOVIS
Males Females Males Females males Females males mOUSE BLOOD
K* □ •6 1-1 (1.6) 1-1 1-2 2-7 1-5 44 (0*1) 4*5
U‘m (1427) (1796)
\l max 1550 120b 4554 2147 208B 1212 1702 2740
HCo 1-20 0.96 1-73 1-46 1-5 1.4 0-75 0.8
K* and W'm denote the values from Hill or Woolf (Hanes) plots (the latter are shown in parentheses).
HCo = Hill Co-efficient.
K' is expressed in [#1 and V'm and V/max are expressed as nmoles product formed/min/mg protein respect
ively.
Table 19
ATP inhibition and FBP activation of the pyruvate kinases from
male and female S.mansoni
ACTIVITY
EFFECTOR CONCENTRATION (uM) HALES FEMALES
ATP
FBP
0 1027
1 x 10"3 1045
1 810
1 x 103 625
0 255
1 239
12.5 465
25 580
50 520
125 510
+ 22 (6) 460 + 25 (6)
± 48 (6) 450 + 0 (6)
± 14 (6) 365 + 17 (6)
± 10 (6) 386 ± 14 (6)
+ 16 (6) 130 + 11 (6)
+ 26 (6) 280 + 63 (6)
± 8 (6) 275 ± 55 (6)
+ 14 (6) 383 + 23 (6)
± 9 (6) 586 + 71 (6)
+ 5 (6) 572 + 32 (6)
Enzyme activity is expressed as nmoles product formed/min/mg
protein + standard error. Figures in parentheses represent the
number of determinations.The PEP concentrations used in the presence of ATP and FBP were 1mM and 0*1 mM respectively.
Table 20
ATP inhibition and FBP activation of the pyruvate kinases from
male and female S.marqreboudei«
ACTIVITY
EFFECTOR CONCENTRATION (uM) HALES FEMALES
ATP
FBP
0 772 ± 13 (A) 297 + 17 (A)1 x 10-3 749 + 2 (4) 309 + 14 (4)
1 546 ± 59 (4) 274 + 26 (4)
1 x 1Q3 3 9 9 + 1 2 (A) 161 + 7 (A)
0 5 7 + 4 (4) 52 CD+1
0*001 ■7A ± 10 (A) 57 1 + CD -O
1 719 ± 23 (A) 85 1 +
1000 2044 ± 25 (4) 1357 + 86 (4)
Enzyme activity is expressed as nmoles product formed/min/mg protein
+ standard error. Figures in parentheses represent the number of
determinations.
The PEP concentrations used in the presence of ATP and FBP were
1mM and 0*1mM respectively.
123
*
♦
4
3.11.5. Interspecific comparisons
All the enzymes studied exhibited positively co-operative
characteristics with the exception of those from female S.mansoni and male S.bovis (Table 18). The PK from male S.marqrebowiei was the most co-operative and active enzyme, although the PK from
male S.mansoni showed the greatest affinity for PEP. The maximum
specific activities of the female enzymes were lower than the males for each species examined.
3.11.6. PYRUVATE KINASE ACTIVITY UNDER "PHYSIOLOGICAL" SUBSTRATE
AND MODULATOR CONCENTRATIONS.
The approximate "physiological” levels of FBP, PEP, ATP and
ADP in both series of S.mansoni were calculated using the metabolite
data given in section 3.9.2. One gram of parasite material was
considered to be equivalent to 1ml of water, thus allowing the in vivo molarity of the metabolites to be calculated.
The K' and V' values for male and female PK were calculated
from Lineweaver/Burk transformations (Conn & Stumpf, 1976) as follows:-
1. Absence of activator/inhibitor : Intercept X axis = - 1Km
2. Competitive inhibition
Intercept Y axis = 1V max
Intercept X axis = ~VKm
Gradient
i-----II ^'hh*'—’ -H
E+
IIITT__
1
KiVmax
[i] = inhibitor concentration Ki = inhibitor constant
3. Uncompetitive inhibition : Intercept X axis = /Km
Intercept Y axis = 1Vmax
The increase in Umax due to FBP was measured using the protocol for competitive inhibition.
3.11.6.1. Enzyme activity in parasite gut contents
It was not possible to quantify the volume of gut contents
present in each sample using the method described in section
2.9.3. The results showed that activity due to gut contents was
low (Table 21). Therefore, only minor contamination of parasite
enzymes by host enzymes will occur.
3.11.6.2. Kinetics of PK from male S.mansoni
Pk activity from male S.mansoni was linear with respect to PEP concentration only under -FBP, -ATP or +FBP, - ATP assay
conditions (Fig. 39). No linear or logarithmic relationship could be established under -FBP, +ATP or +FBP, +ATP conditions.
Lineweaver/Burk transformations were carried out to elucidate
the effects of FBP and ATP on unmodulated enzyme activity (Fig. 41).
; j*Ki
1 +JSLKi
Table 21
Enzyme activity in gut contents of female S.mansoni.
ENZYME ACTIVITY
Pyruvate kinaseGlucose-6-phosphate dehydrogenase
Malate dehydrogenase (MAL-OXO)
Citrate synthase
2 + 0.01 (5)
□•1 + 0-01 (4)
0*02 + 0*001 (4)
n.d.
n.d. = activity not detectable.
Results are expressed in nmoles product formed/min/mg protein
+ standard error. Figures in parentheses represent the number
of determinations
Regression plots, K! and Vfm values produced by -FBP, -ATP
and -FBP, +ATP conditions, did not fit any of the standard inhi
bitor classes i.e. non, un or competitive inhibition (Fig. 41,
Table 22) (Lehninger, 1977). However, statistical comparison of mean activities at each PEP concentration showed that the
addition of ATP produced an overall significant (Pc:0*02) 37% reduction in enzyme activity. +FBP, -ATP and +FBP, +ATP
conditions resulted in regression plots which indicated that ATP
inhibited the erlzyme uncompetitively. In support of this, the calculated values of K* and V'm showed a large decline (Table
21) and subsequent statistical analysis of the untransformed data (Fig. 39) showed a significant (P<0*02) overall 43% re
duction in enzyme activity. The presence of FBP raised the V*m
of the enzyme (Fig. 41, Table 22). This was confirmed by
analysis of the untransformed data (Fig. 39) which showed a significant (P<0*05) 69% increase in activity.
3.11.6.3. Kinetics of PK from female S.mansoni
PK activity in female S.mansoni was linear with respect to PEP concentration under all assay conditions (Fig. 40). The
data was transformed to Lineweaver/Burk plots which were subse
quently fitted with regression lines (Fig. 42). Comparison of PK activity under -FBP, -ATP and -FBP, +ATP, indicated that ATP
exerted an uncompetitive inhibitory effect. However, statistical
comparison of the untransformed data (Fig. 40) showed that ATP did not significantly inhibit PK activity. Comparison of PK
Table 22
Apparent Km (K')» Vmax (V!m) and inhibitor constant (Kl) for the pyruvate kinases from male and female 5«mansoni.
♦ # * *
MALES FEMALES
MODULATOR K' (pM) \ltm KI (pM) k * (urn) V'm KI (pKl)
- FBP - ATP 61 201 28 153
- FBP + ATP 99 475 0.1 22 88 769
+ FBP - ATP 704 1504 20 195
+ FBP + ATP 39 11 90 72
K' = apparent Km.
W'm = apparent maximum velocity (nmoles product formed/min/mg protein).
KI = inhibitor constant.
Figure 41
Lineiueaver/Burk plot of the effect of "physiological" levels of PEP (10 - 50pM), ADP (0-5r#l), FBP (23pl*l) and ATP (2*25mM)
on the activity of pyruvate kinase from male S.mansoni.
Figure 42
Lineueaver/Burk plot of the effect of "physiological" levels of PEP (10 - 50|j|Yl), ADP (0-4r#l), FBP (50pl>l) and ATP (0-6r#l)
on the activity of pyruvate kinase from female S.mansoni.
Fig. 41
■ -F B P -A TP o — FBP+ATP a +FBP—ATP • +FBP+ATP
Fig. 42
b _FBP—ATP o —FBP+ATP a +FBP-ATP *+FBP+ATP
[PEP] pM
Figure 39
The effect of "physiological" levels of PEP (10 - 50^),ADP (O'SmM), FBP (23^) and ATP (2-25mM) on the activity of
pyruvate kinase from male S.mansoni.The values represent the means of 5 determinations +
standard error.
Figure 40
The effect of "physiological" levels of PEP (10 - SOjjM),
ADP (0*4mfl), FBP (50^) and ATP (O'BmM) on the activity of
pyruvate kinase from female S.mansoni.
The values represent the means of 5 determinations +
standard error.
Activ
ity(n
mol
es/m
in/m
g pr
otei
n)
Activ
ity(n
mol
es/m
in/m
g pr
otei
n■ —FBP—A TP o—FBP+ATP a +FBP-ATP • +FBP+ATP
■ — FBP—ATP o -FBP+ATP a +FBP-ATP • +FBP+ATP -
behaviour under +FBP, -ATP and +FBP, +ATP conditions indicated
that competitive inhibition was occurring (Fig. 42). Although
the K! increased, the V'm decreased (Table 22) indicating that
this effect does not strictly fit the standard inhibitor classes
(Lehninger, 1977). Analysis of the untransformed data (Fig. 40)
showed that PK activity was significantly reduced (P<=0*02)
overall by 33?.The addition of FBP alone (+FBP, -ATP) showed that the K ’
was lowered and the M ' m was increased (Fig. 42, Table 22). This
indicates that enzyme activity was increased and statistical analysis of the untransformed data (Fig. 40) showed that activity
was significantly enhanced (P<G»05) overall by 22?.
PK activity in male S.mansoni was significantly (P<0«02)
higher than in females by an average of 65? at all PEP concen
trations except lOpM. At this concentration there was no sig
nificant difference in activity. In the presence of FBP, male
PK was eight times more active than the female enzyme. Male PK
in the absence of FBP, was more significantly (P<0*001) inhi
bited by ATP than the female enzyme. In the presence of FBP the
enzymes from both sexes were inhibited by ATP, to the same degree.
3.11.6.4. Stability and the effect of cations on the activity of PK
The activity of male and female PK in the absence of FBP
and ATP showed no significant changes over 7h- (Fig. 43). Male and female PKs showed no significant alterations in activity when assayed in the presence of Mg’ or Mn" (Figs, 44 & 45).
Figure A3
Stability of pyruvate kinase from mal-e and female S.mansoni
over a 7h period. [PEP] = 50jjm
The values represent the mean of 5 determinations + standarderror
Fig.43
Figure 44
The effect of Mg and Mn on the activity of pyruvate kinase
from male S.mansoni.
The values represent the means of 5 determinations ± standard
error. The concentration of cation used was 5mM.
Figure 45
The effect of Mg and Mn on the activity of pyruvate kinase from female S.mansoni.
The values represent the means of 5 determinations + standard
error. The concentration of cation used was 5mM.
*
Activity(nmoles/min/mg protein)r—*
CJ1 O? ________£
vx‘-fc*cn
*-0S
I
lAfri
§13d
]
* *
Activity(nmoles/min/mg protein)i-j. |\i CJo o o
Fig. 44
3.11.6.5. Pyruvate kinase from mouse erythrocytes
Using the technique outlined in section 2.9.4. it provedfeimpossible^obtain a serum sample that was free of haemoglobin.
As other intracellular components may have leaked into the serum
fraction, only the data for mouse erythrocyte enzymes were recorded.
In the case of erythrocyte PK, insufficient data was available to
determine the shape of the graph at lower levels of PEP concentration. Therefore it was not possible to determine whether the
enzyme oo«sexhibiting Michaelis-Menten or sigmoidal kinetics (Fig.46).
The K ’ and HCo for the enzyme are shown in Table 18.
Glucose-6-phosphate dehydrogenase (1955 + 64 nmol/min/ml)
and malate dehydrogenase ( 8 8 + 3 nmol/min/ml) activity was found
in erythrocyte samples but citrate synthase was not detectable.
3.12. ELECTROPHORESIS
Experiments with polyacrylamide gels were not successful as
many samples failed to migrate into the resolving gel. The PK from mouse erythrocytes produced a single band (Plate 1). Starch gels were more productive as two PK isoenzymes consisting of one major and one minor staining band were detected in male and fe
male S.mansoni (Plate 2). In the females, the minor staining
isoenzyme took longer to develop than that from the males. In
the case of cellulose-acetate gels, parasite samples produced
a single major staining band for PK as did the enzyme from mouse
blood (Plate 3).
Figure 46
The effect of PEP concentration on the activity of pyruvate
kinase from mouse erythrocytes.
The values represent the means of 4 determinations + standarderror
♦
Plate 1
Isoenzyme pattern of mouse erythrocyte pyruvate kinase using
polyacrylamide gel electrophoresis.
Plate 2
Isoenzyme pattern of pyruvate kinase from
male and female S.mansoni using starch gel electrophoresis.
Plate 3
Isoenzyme pattern of pyruvate kinase from mouse erythrocytes and
male and female S.mansoni using cellulose-acetate electrophoresis.
Plate.1
Plate.2
Plate.3
Haemoglobin
Erythrocyte PK
Plate 4
Isofocusing patterns of pyruvate kinase from male and female
S.mansoni.
Plate.4
3*8-i
4-3- 4*G“
p H 4-9-
6-2-
7 0 J
cf $ Haemoglobicontrol
3.12.1 Isoelectric focusingIsoelectric focusing rev/ealed that 6 similar isoenzyme bands
were produced by both male and female S.mansoni (Plate 4). 5pl aliquots produced the clearest patterns and these corresponded
to 50pg and 30|jg of protein for male and female samples respect
ively. One major band of activity, with an isoelectric point of
6*2 was evident in both samples and these bands developed at the
same rate.
3.13. CYTOCHROMES
Quantitative calculations of cytochrome levels were perfor
med using the wavelength pair data of Wilson & Epel (1968)
according to the following equation:-
0.D.1 - 0.D.2 x \I = mmoles/ml sample (E) ( U U.F.)(u)
O.D.1/2 = optical density at specific wavelengths (nmetres).
\J = Cuvette volume
E = Extinction co-efficient.
L = Light path (cm).I.F.= Intensification factor (7) (Wilson, 1967).
v = Sample volume.
3.13.1. CONTROLS
3.13.1.1. Whole mouse blood and rat liver
Three minor peaks were found in the citrated blood samples
at 454, 530 and 575 nm (Fig, 47). The scans obtained from rat
liver showed peaks at the expected wavelengths for cytochromes a, a^» c, c and b i.e. 602, 445, 550, 556 and 564 respectively
(Hayes, 1983) (Fig. 48).
3.13.1.2. Digested mouse bloodThe scans of pepsinised mouse blood are shown in Fig. 49.
The areas of scans that overlap, represent the wavelengths that
would be cancelled out in the spectrophotometer during assay i.e. those wavelengths that would not be recorded as an increase
or decrease in absorbance. Therefore it is evident that the
presence of blood digestion products will interfere significantly with any scans between 484 - 680 nm.
3.13.1.3. HaematinThe scans of the haematin solution are shown in Fig. 50.
These results show that there are very slight differences in absorbance between the reduced and oxidised states from 400 -
497 nm and 647 - 700 nm. However, between 497 - 647 nm large differences in absorbance occurred. Therefore, haematin
represents a comparatively larger source of contamination than the digested blood preparation, particularly in the range cover
ing cytochromes c» Oj and b i.e. 550, 556 and 564 nm respectively
3.13.2. S.MANS0NI
Figure 47
Cytochrome scan of whole mouse blood.
Figure 48
Cytochrome scan of rat liver
Abso
rban
ce
Fig. 47
1-1
*
*
454I .....i “ "
530 575
Wavelength(nm)
4-
*•
Fig. 48
556 5 6 4
Wavelength(nm)
Figure 49
Absorbance scan of pepsinised mouse blood.
Abso
rban
ce
* * lyif -(jifc. 9*
Figure 50 4
Absorbance scan of a solution of haematin.
*
4
4
m
Abso
rban
ce
Wavelength(nm)
+ ¥ i I* &-
3.13.2.1. MalesCytochrome peaks were detected at 445, 548, 556 and 564
nm and comparison with Fig. 48 shows these are cytochromes a^»
c, c and b respectively (Figs, 51 & 53). Haematin, although
present, did not prevent the peaks from being recorded. No
peak was recorded at 602 nm suggesting that cytochrome a was not
present but a large increase in absorbance was found at 640 nm
in one sample. Subsequent scans of several different samples
from 600 - 700 nm failed to show similar peaks, so it was
assumed that the first sample included an unknown artefact. Quantitative data for the cytochromes are shown in Table 23 and cytochrome c is the most abundant.
3.13.2.2. FemalesPeaks were detected at 445, 548 and 566 nm thus indicating
the presence of cytochromes a^, c and c^, (Fig. 52). No peaks
were found at 564 nm even in the presence of antimycin A and
succinate, indicating that cytochrome b was not present or levels
were too low to be detected. As with the males, a large peak
was found at 640 nm in one sample but not in others, hence, as
for the male sample, this was presumed to be an artefact. Cytochrome a^ levels were the most abundant, being three times higher
than in the males (Table 23) but levels of cytochromes c and c
were approximately the same in both sexes.
3.14. OXYGEN UPTAKEMale and female S.mansoni both took up oxygen (O2) from the
Figure 51
Cytochrome scan of male S.mansoni
Figure 52
Cytochrome scan of female S.mansoni
Full
scal
e ab
sorb
ance
Fu
ll sc
ale
abso
rban
ce
Fig.51
Fig. 52
Wavelength(nm)
Figure 53
Absorbance scan of cytochrome b from male S.mansoni.
00 p # 0
Full scale absorbance
Table 23
Cytochrome levels in male and female S.mansoni.
CYTOCHROME MALES FEMALES
a 30*06 + 0*001 (6) 0-2 + 0*004 (5)
c 0*14 ± 0*004 (5) 0.1 ± 0*01 (5)
C1 0*07 + 0*001 (5) 0*1 + 0*002 (4)b 0*02 + 0*001 w n.d.
n.d. = not detectable.
Results are expressed as nmoles/mg protein + standard error.
Figures in parentheses represent the number of determinations.
medium but the females took up 8 times as much 02 as the males
(Table 24). The agitation necessary to maintain hyperbaric
conditions did not appear to visibly damage the worms. Males
and females were found to be motile after 30 min, prior to the
addition of KCN. After this point, there was a large decline in the motility of both sexes. O2 uptake by male S.mansoni was inhibited completely by KCN but uptake by the females was only
inhibited by 64%
Table 24
Oxygen uptake by male and female S.mansoni.
MALES FEMALES
- KCN 2 + 0*1 (5) 16 ± 1 (5)
+ KCN 0.0 (5) 5.8 + 0«3 (5)
Results are expressed in jjl 0 ^ consumed/h/mg protein
standard error. Figures in parentheses represent the
number of determinations
CHAPTER 4
#
0
0
DISCUSSION
4.1. GLUCOSE, GLYCOGEN, PROTEIN, WET WEIGHT AND LIPID LEVELS
Both sexes of S.mansoni and S.marqrebouiiei maintained fairly constant levels of glucose between 42 - 84 days post infection.
Glucose was measured relative to protein levels which did change
over the time course of measurement. These alterations were small
and do not affect the overall relationships between glucose levels
in the different species. This highlights the potential problems
of relating metabolite levels to a baseline which changes with time
without assessing the changes. Particular difficulties might be
encountered when comparing values obtained with the data of other workers. Different worm burdens, the species, strain and nutritional status of the host may significantly affect parasite consti
tuents hence making reliable comparisons difficult.The major problem with using wet weight as a baseline is the
difficulty encountered in removing all extracellular water from the parasite (Radke et al, 1957). In addition, the contribution of
parasite gut contents to the measurements would be extremely difficult to account for. Presumably, the gut contents are likely to
cause greater variation with female measurements as these generally
contain more digested blood than the males. Howeveiv the latter
could also retain additional water in the gynaecophoric canal. Dry
weight measurements of samples would serve as a more reliable base-,
line but would preclude the determination of labile components in
the same sample. Expression and comparison of quantities on a
single worm basis are also beset by the same problems. Therefore
the use of protein as a baseline appears to be the most reliable
means of comparing the data of different researchers.
Glycogen levels are higher than those for glucose in both S.mansoni and S.marqrebowiei. These levels remain stable over the
6 week measurement period with the exception of male S.marqrebowiei.
These observations are in keeping with the findings of Bueding & Koletsky (1950). Male levels of glycogen were higher than those of
the females in both species which is also in agreement with the work
of Bueding (1950). As male worms are more mechanically active in vivo
than the females, the higher levels of glycogen are probably essential to meet energy demands. The larger glycogen stores in
the males may also be accessible to the female parasites via a glucose
transfer mechanism (Cornford & Huot, 1981). This may be another aspect of the nutritional dependance of the female on the male
(Senft, 1968) and indicates a division of metabolic labour between the functional schistosome pair.
Smyth (1966) proposed that the best means for assessing tissue
growth is on a weight basis. Therefor^ the present study indicates
that S.mansoni had reached a stable growth level by 42 days post infection, whereas S.marqrebowiei continued to grow after this time.
Female S.marqrebowiei appear to plateau off at about 56 days but
the males increased their weight up to 84 days post infection. This
increase may be linked with the synthesis and storage of glycogen
in this parasite mentioned earlier. Very little is known concerning glycogen synthesis in schistosomes, therefore male S.marqrebowiei may serve as a useful model for future studies in this area.
Cornford et al (1982) found that protein levels in female S.mansoni declined after 98 days post infection. This is thought
to coincide with the chronic phase of infection (Cheever et al, 1980)
and hence the fall in protein levels is thought to be linked with the host^ immune response. In the present study, all but male
S.mansoni showed a decline in protein levels so indicating a possible
response to host immune attack by 56 - 70 days post infection. It
should be noted that higher numbers of cercariae were used for in
fection in the present study than used by Cornford et al (1982).
Therefore, this may have elicited a more rapid host response.
Wilson & Barnes (1977) found a high rate of membrane protein
turnover in S.mansoni and as tegument renewal is thought to be a part of an immune evasion mechanism, more protein may be diverted
to the tegument and subsequently shed. This would account for the decline in parasite protein levels. The quantity of protein per
worm in S.mansoni in the current investigation shows close agree
ment with the values found by Cornford et al (1982) and Watts (1978).
Male S.mansoni contain higher levels of total lipid/mg protein
than female worms. However, if these values are related to the wet weight of the parasites then the levels in males and females
are similar i.e. 11% and 9% respectively. Lipid in the female parasite probably plays a significant role in egg production
(Erasmus, 1973) but its function in males is less clear. Lipid is
also a major component of the schistosome tegument (Smith & Brooks,
1969) and so, relatively high levels would have to be maintained as
the parasites have a double plasmalemma and the rate of tegumental
turnover is high (Wilson & Barnes, 1977).
4.2. ENZYME ANALYSIS
Measurement of the specific activities of enzymes involved in
different metabolic pathways provides a rapid means for a broad comparison between species and affords a general view of which path
ways are predominant. The main drawback with this type of analysis
is the use of standardised assays involving saturating levels of
reactants and co-factors, which do not account for the individual
optimal requirements of each enzyme.
The results of the present study indicate that glycolysis is
a major source of ATP generation in schistosomes and so agree with
the findings of Bueding (1950) and Shapiro and Talalay (1982b). A possible exception to this was S.haematobium (S.African strain) as
the maximal L-lactate dehydrogenase (L-LDH) activities were low in
comparison with other species. The incubation of 5.haematobium (Morroccan strain) also suggested that glucose can also be meta
bolised via a non-glycolytic pathway.liiaitz (1964), Bruce et al (1974), Coles (1973) and Huang (1980)
have produced evidence to suggest that S.mansoni and S.japonicum
are capable of operating a tricarboxylic acid (TCA) cycle and
hexosemonophosphate (HMP) shunt.The enzyme activities measured in the present study indicate
that the pathways, if operational, are running at a low rate. There
fore; their contribution to ATP synthesis may be at best, relatively
small. All TCA enzymes were detectable in S.marqrebowiei but only
citrate synthase, aconitase, and malate dehydrogenase (MDH, oxidising)
were measurable in S.mansoni, suggesting that the TCA cycle may not
be functional in the latter. The significance of the suggestion is
more fully discussed in the incubation section (4.4). It should be noted that the addition of Triton X-100 to samples showed that
normal homogenisation was only liberating about 50% of the TCA
enzymes. Therefore, activities are likely to be approximately twice
the recorded values.The enzyme data for S.mansoni shows close agreement with the
results of Coles (1973b) and Shapiro & Talalay (1982b) although there are a few notable differences. Shapiro & Talalay (1982b) found the
activity of hexokinase (HK) in S.mansoni to be low and suggested
that it may be the main regulatory enzyme of glycolysis. The HK
activities of S.mansoni and S.marorebowiei recorded in the present
study were much higher. Therefore, the enzyme phosphofructokinase
(PFK) may be playing a more important role in regulation.
Phosphoenolpyruvate carboxykinase (PEPCK) activity was also
lower in both species than the values obtained by Bueding & Saz (1968)
and Coles (1973b). It is unfortunate that this enzyme may be subject to pyruvate kinase (PK) interference as this could explain the
variation. The presence of nucleoside diphosphokinase activity in
S.mansoni supports PEPCK activity in the direction of CO^ fixation
(Zammitt & Newsholme, 1976). However, the relative specific
activities of PEPCK and PK suggest that very little phosphoenol-
pyruvate (PEP) participates in CO2 fixation in either species. In
support of this, the failure to detect fumarase suggests that CC^ fixation and a reverse TCA cycle do not operate in S.mansoni.
Shapiro 4 Talalay (1982b) suggested that the presence of MDH
and glycerol-3-phosphate dehydrogenase (GPDH) in S.mansoni indicated
the possible existence of a functional electron shuttle system. The
present study revealed that all species studied showed MDH and GPDH
activity, the latter being particularly active in S.mansoni. The
significance of this high activity and the possible role of MDH are more fully discussed later (section 4.4).
The presence of glucose-6-phosphate dehydrogenase (G-6-PDH)
and 6-phosphogluconate dehydrogenase (6-PGDH) is usually acknow
ledged as indicating the presence of a functional HMP cycle and these enzymes were active in each species examined. In addition, transal
dolase activity was found in S.mansoni but Barrett (1981) points out
that the pathway may terminate at ribulose-5-phosphate formation.
Although U/aitz (1964) has found evidence to suggest that the cycle
is functional in S.mansoni, further work on this area of metabolism
may prove interesting as the cycleTs role as an anaplerotic mech
anism and a source of synthetic molecules has not yet been evaluated.
154
4.3. METABOLITES
#-The difference in mouse liver metabolite levels found between
the data from the present study and Williamson & Brosnan (1974)
may be attributed to the use of different anaesthetics used to
#■ . kill the animals. Killing without anaesthetics can also influence
the results as it may cause the tissue to respond so that it is not
in a resting state. Dickerson (1965) found that anaethesia pro
duced a hepatic shift in schistosomes in mice, thus it is possible
that parasite metabolites will also be influenced by whichever
method is used to kill the host.L-lactate was the most abundant intermediary metabolite in
* S.mansoni and 5.marprebowiei. This supports the idea that schistosomes are heavily dependant on glycolysis as a source of
energy. Oxaloacetate (OAA) levels were also relatively high in
^ males of both species but the metabolic route of formation in theseparasites has yet to be demonstrated. The possible implications of
the high levels of this metabolite are discussed more fully in section
4.4. The inability to detect other TCA cycle intermediates again
suggests that the cycle, if operational, is running at a low rate
and so would not contribute much to ATP synthesis.Dihydroxyacetone phosphate (DHAP) was detected only in S.mansoni
and this is probably due to the activity of glycerol-3-phosphate dehydrogenase (GPDH) which was particularly high in this species.The DHAP could be a precursory step in de novo neutral lipid synthesis or could be participating in a mitochondrial shuttle
AI55
#
*
%
The general levels of metabolites are lower than reported for other parasites eg Hymenolepis (Rahman & Mettrick, 1982) Echinococcus
(McManus & Smyth, 1982) Ligula (Sterry, 1980). Yet the values
do fall within the ranges reported for other invertebrates (Barrett
& Butterworth, 1982), (Beis & Barrett, 1979) and (Beis & Newsholme, 1975). Differences between the data of different workers may be
due to variations in protocols such as methods of wet weight determination or the time taken to recover and freeze the parasites.
The ATP level in male S.mansoni is very close to the value
reported by Bueding & Fisher (1982) for paired worms. The adenylate
charges for S.mansoni indicate that the male and female worms were
in a normal, healthy state prior to removal from the host(s)
(Atkinson, 1971). The higher ATP : AMP ratio and level of ATP
in the male worms indicate that the rate of energy utilisation and
the rate of change of the glycolytic flux is higher than in the females (Beis & Newsholme, 1975). These points suggest that the
females maintain a steadier rate of glycolysis than the males. This may be viewed as a reflection of their probable in vivo life-style in
that they are presumably less mechanically active than the males, who transport the females through the hosts’ mesenteric/portal/urinary
blood vessels. Conversely, the males would require periodic bursts
of energy expenditure to perform this activity and hence they appear to be able to change rapidly from a resting state of glycolysis to
a more active one. This rate of change may be mediated by a marked
sensitivity to FBP which is discussed later (section 4.5.).
system, coupling to a cytochrome electron transport chain.
155
♦
♦
4
4.4. INCUBATIONS
Great care should be exercised when extrapolating the results of in vitro experiments to build up a picture of in vivo biochemistry
as most incubation conditions do not provide an optimal environment for normal metabolism. Variations in media constituents, gaseous phase and the duration of incubations could possibly exert
great influence on the type and quantity of metabolites taken up,
metabolised and excreted/secreted by the parasites. Additionally,
schistosomes contain quantities of host enzymes within their digestive
tracts which could affect assays. However, the assay of certain
gut enzymes in the present study suggests that interference from host enzymes is minimal. This supported by the detection of only
small quantities of radiolabelled CC^ produced by mouse blood during
incubation.L-lactate was the major detectable end-product of glucose
catabolism in each species investigated and this supports the earlier evidence that glycolysis is a major contributor to ATP syn
thesis in these parasites. For the males of each species examined
except 5..haematobium, lactate production stoichiometrically balanced glucose consumption. Thus it seems reasonable to describe these
organisms as essentially lactate fermenters and so.supports the findings of Bueding (1950), Schiller et al (1975) and Shapiro &
Talalay (1982b). In contrast, glucose consumption in S.haematobium exceeded lactate production, indicating that a proportion of glucose must be catabolised via a non-glycolytic pathway. Male S.mansoni
and S.marqrebowiei both produced low levels of CO^ and the former
contained small quantities of glycerol which suggests that alter
native pathways of glucose catabolism are operational.
Bruce et ad (1974), Coles (1973), Huang (1980), Smithers et al
(1965) and liiaitz (1964) presented evidence that S.mansoni and
5.japonicum possess functional tricarboxylic acid (TCA) and hexose- monophosphate (HMP) pathways which may be the mechanisms of CC^ production by male S.mansoni, S.marqrebowiei and S.douthitti.
S.marqrebowiei was found to have a full complement of TCA enzymes and the first two enzymes of the HMP pathway thus suggesting that these
cycles are operational in this species. Male S.mansoni only
exhibited citrate synthase, aconitase and malate dehydrogenase
(MDH, oxidising) activity and the only TCA metabolite detectable
was oxaloacetate (OAA). Therefore, in S.mansoni, the TCA cycle may
not be functioning and CC^ production might be due to HMP shunt
activity or amino acid breakdown (Senft, 1968), (Bruce et al, 1972).
This poses the question as to how OAA is formed by the parasite and
more importantly, what its function is. In both 5,mansoni and
5marqrebowiei OAA levels were higher in males than females. OAA
was not secreted/excreted in appreciable quantities so presumably it is an intermediate metabolite.
If the TCA cycle is functioning, part of the OAA produced would
be condensed with acetyl coenzyme A(ACoA) by citrate synthase in
order to continue the cycle. However, if this is the case, then there would appear to be some sort of restriction of citrate synthase
activity as the data (Tables 15 & 16) shows there is a build up of
□AA. Availability of ACoA could provide a means of limiting citrate
synthase activity. As no pyruvate dehydrogenase (PDH) activity
was detected in S.mansoni the ACoA would have to be derived from
another source eg amino acid or lipid breakdown.The OAA could be an essential component of a transmitochondrial
membrane electron shuttle system. MDH and glutamate-oxaloacetate transaminase (GOT) activity were detected in S,mansoni and these
enzymes could channel aspartate into the cytoplasm. Phosphoenol-
pyruvate carboxykinase (PEPCK) and MDH would provide the malate which
carries electrons into the mitochondria (Lehninger, 1977). A pos
sible function of the shuttle system may be to furnish electrons for
the cytochromes detected in S.mansoni in the present study (Section
3.13). Transaminase activity was also reported by Senft (1963) and
the possibility exists that OAA may be a precursor for amino acid synthesis. Therefore, OAA could possibly be fulfilling a general utility role in metabolism.
It is unfortunate that much of this speculation is dependent
on the cellular distribution of the enzymes involved, of which little
is known. Rotmans (1978) reported that cytoplasmic and mitochondrial MDH showed a twelve fold higher activity for OAA reduction than for
malate oxidation. In addition, the mitochondrial enzyme was found
to be more active than the cytoplasmic form. If this is so then
there would tend to be a build-up of malate in the mitochondrion.
The malate may pass into the cytoplasm for subsequent metabolism by MDH. This mechanism could play a part in providing intramitochondrial NAD+, which in turn could assist in the formation of 2-oxoglutarate
and succinyl coenzyme A. Overall, the proposed MDH activity u/ould
hamper TCA operation unless regulated by some means. Also it may
tend to obstruct the involvement of cytochromes. Therefore it would
probably be more prudent to bear in mind that Rotmans* findings were
for partially purified samples studied under non-physiological
conditions and so the data produced may not be applicable to the
in vivo situation.If the TCA cycle is not operating in S.mansoni then the enzymes
detected may be relics of a redundant cycle. Shutdown of the TCA
cycle may be caused by a decline in availability of ACoA, mediated
via PDH activity. It would be interesting to monitor the activity of this enzyme in cercariae and schistosomulae to determine if there
is a change in activity with development. Citrate synthase, aconitase
and MDH could be participating in a glyoxylate cycle which could
contribute to OAA synthesis but it would require a source of ACoA.
However, the presence of isocitrate lyase and malate synthase has
not been demonstrated in schistosomes. If citrate is not being
produced by S.mansoni in vivo, this may account in part for the apparent insensitivity of phosphofructokinase to this metabolite
(Bueding & Fisher, 1966).As mentioned previously, cytoplasmic PEPCK in male S.mansoni
and S.marqebowiei could catalyse the formation of QAA which could
further be reduced to malate by MDH. PEPCK activity in S.mansoni may be augmented by the action of nucleoside-51-diphosphokinase (NDP)
which would provide inosine -5*-triphosphate (ITP). However, there
150
♦
♦
is no evidence to support the idea that subsequent metabolism of malate to pyruvate, acetate, alanine or succinate occurs in either species. Also, the relative activities of pyruvate kinase (PK) and
PEPCK would severely limit the amount of phosphoenolpyruvate (PEP) available for fixation. Therefore it seems more likely that
PEPCK may be fulfilling a more anabolic role via gluconeogenesis.
Whatever the physiological role or means of formation of OAA, the
relative levels indicate that it is more important in the metabolism of male parasites.
In view of the available data, it is not known conclusively
whether the strain of male S.mansoni used forrthe present study
possesses fully functional TCA and HMP pathways. However, the evidence does suggest that 5 jnarqrebowiei is capable of at least
operating a TCA cycle. The contribution of the TCA cycle in each species to energy production appears to be minimal in both species.
The female schistosomes studied consumed far more glucose
than could be accounted for by lactate production. L-lactate was
the major end-product detectable and was produced in similar quan
tities as for the males. This suggests that the average rate of
lactate production during incubation was the same for male and female
worms. This contrasts with the work of Bueding & Fisher (1982) which
showed that males had a higher rate of glycolysis over 2h and 18h
incubations. The difference in results could be due to the different media, protocols or parasite strains used. The extra glucose con
sumed by the female parasites could possibly have been catabolised by a non-glycolytic pathway or used in an anabolic process.
OAA may fulfil similar roles in the biochemistry of the female
parasites as previously suggested for the males. The sex related
difference in OAA levels may be linked with the females' apparent
metabolic dependence on the males (Lennox & Schiller, 1972). If OAA is fulfilling the role of a multipurpose metabolite, then the
males could be acting as a store for the female parasites. Cornford
& Huot (1981) observed a similar phenomenon for glucose.As lactate u/as the only major end-product detectable in female
incubates then it seems possible that the extra glucose consumed
by the females may have been used for an anabolic purpose and the
most likely fate would be egg production. S.mansoni and S.marqrebowiei produce an average of 118 (Schiller et al, 1975) and 837 (Southgate
& Knowles, 1977) eggs per day. Sauer & Senft (1972) estimated that
daily egg production by S.mansoni is approximately equivalent to one
tenth of the females' dry body weight. Therefore, egg production
would appear to be an intensely metabolic process and probably requires
large quantities of ATP. This is supported by the fact that S.mansoni oocytes are surrounded by large numbers'of mitochondria (Roberts, 1983). Additionally, the miracidium is thought to be solely dependent on
glycogen as an energy source (Coles, 1973) and so this would probably
be synthesised by the female worm. Smith et al (1971) suggests that free fatty acids may also act as a source of miracidial energy but
it is currently thought that the female worm is not capable of de novo fatty acid synthesis (Meyer et al, 1970). Therefore the excess
glucose could be used to furnish energy and energy storage compounds for the egg and miracidium.
Egg production also requires a source of amino acids for egg
shell formation. Female S.mansoni metabolise several amino acids
(Bruce et al, 1972) and Senft (1963) reported that 6$ of glucose
taken up by worms is converted to alanine. Chappell & Walker (1982) found that glucose did not represent a significant source of protein
amino acids except for alanine, aspartate and glutamate. These
amino acids were found to be incorporated into worm tissue and egg
proteins. In the present study, alanine was not detected in worm
homogenates possibly as the quantities present were outside the
sensitivity range of the assay used. Nevertheless, a small quantity
of glucose was found to have been incorporated into parasite tissue
(Fig. 20). Haemoglobin degradation by the gut proteolytic enzyme may not furnish sufficient amounts of free amino acids (Saver & Senft, 1972) and thus some mechanism of synthesis from other compounds such
as glucose may be of great importance to the parasite. Thus, a
portion of the consumed glucose may go to form structural components for the adult parasites and for egg formation.
Little is known concerning general lipid metabolism in schisto
somes. Meyer et al (1970) reported that paired S.mansoni were incap
able of d£ novo synthesis of saturated fatty acids but retained the
ability to manufacture complex lipids from exogenous sources of fatty
acids and sterols. Venous blood could presumably act as a source of
these lipids. Lipid is a major constituent of schistosomes, being
one third of their dry weight (Huang, 1981). Smith & Brooks (1969) found that free sterols and triglycerides are the major neutral lipid
fractions in S.mansoni and Fried et al (1981) reported that S.mansoni
released free sterols, fatty acids, di and triglycerides into an
incubation medium within 2h. These products may simply be the
result of a desaturation and breakdown mechanism (Meyer et al, 1970).
Yet it is interesting to note that both sexes of S.mansoni contained
small quantities of glycerol. This could have been of host origin
but does not help to explain the relatively high levels of glycerol-
3-phosphate dehydrogenase (GPDH) activity and dihydroxyacetone phosphate (DHAP) that were found in the present study. GPDH activity
and DHAP levels in male and female S.marqrebowiei were lower and no
glycerol was detected in this species. Shapiro & Talalay (1982b) found a very low activity for GPDH in paired S.mansoni and it may be
possible that the strain of S.mansoni used for the present study is capable of glycerol synthesis. Hence, a proportion of the excess
glucose could be channelled along this route. Glycerol could sub
sequently be linked to fatty acid synthesis which in turn may be
central to the metabolism of vitellogenesis in female worms. GPDH
and DHAP may also be important in terms of electron transfer into
the mitochondria for subsequent coupling to electron transport chains. This may be of particular importance in female S.mansoni as they appear to be more dependent on oxidative phosphorylation than the
males (Section 3.14.).Renewal of the schistosome tegument might be another means by
which the excess glucose could be metabolised. Wilson & Barnes
(1977) reported that the large number of discoid granules present in the tegumental cytoplasm contain mucopolysaccharides. . Hockley
(1973) and Morris & Threadgold (1968) reported that non-glycogenic
PAS positive carbohydrate is present on the surface membranes of
S.mansoni. Wilson & Barnes (1977) also estimated that the schist
osomes tegument has a half-life renewal of 2-3 hours therefore,
glucose incorporated into tegumental macromolecules could be shed
into an incubation medium and so remain undetected. No data on the relative quantities of glucose present in the tegument or relative
membrane turnover rates of male and female worms are presently available. If large amounts of incorporated glucose are present in
the tegument then membrane turnover may account for the apparent disappearance of glucose. Kusel & Mackenzie (1975) also found that
fragments of schistosome tegument were sloughed off during incubation.
If tegumental renewal is a main cause of the glucose discrep
ancy then it follows that the females appear to have a higher turn
over rate than the males. This seems odd, as the males would pre
sumably present a greater surface area for potential immune attack
by the host system. It is apparent the further experiments should
be geared to the quantitative recovery and analysis of shed membrane
fragments on a comparative gender or species basis.Nash et aL (1974) and Deelder et al (1976) reported that schis
tosomes released circulating- antigens which were substantially carbohydrate in nature. The quantity of carbohydrate was estimated to be approximately 1% of the lyophilised worm weight for paired worms. This is another potential outlet for the excess consumed
glucose. The antigens are thought to be released as a means of
evading the host immune response.
165
Differences in the glucose : lactate (G:L) ratios between male and female parasites may simply be a reflection of their potentially
greater anabolic nature. It is difficult to assess the relative contributions made by male and female worms to the integrated
metabolism of the adult pair as it is not known to what extent they
are dependant on one another.
Glycolysis appeared to be the major detectable means of ATP
synthesis in the schistosomes studied and the presence or absence of
a functional TCA cycle in each of these particular species remains
to be confirmed. Evaluation of the energy contribution made by electron transport chains is now essential, particularly the means of
coupling to redox reactions, as this mechanism of energy production
may be of great singificance in the biochemistry of the female parasites. The ultimate fate of glucose, especially in female meta
bolism, appears to be potentially manyfold and therefore these worms should not be regarded as solely homolactic fermenters.
4.5. PYRUVATE KINASE
The study of pyruvate kinase (PK) activity in S.mansoni at
saturating assay conditions, indicates that the male enzyme is
susceptible to regulation by FBP and ATP. The female enzyme, while
activated by FBP, is far less sensitive to inhibition by ATP. This is in direct contrast to the findings of Brazier & Jaffe (1973) who reported that paired S.mansoni PK was insensitive to FBP activation
in the range 0.1 - O^mM and ImM ATP inhibited activity by 10p only
and so they concluded that the enzyme was not regulatory. Clearly,
the enzyme in the present study shows different characteristics and
this may be due to strain variation or to the fact that the male
and female enzymes were examined separately.
The results suggest :that male and female PK may be important
in controlling glycolytic flux but the method of control is different
in each sex. The sigmoidal character of the male enzyme and its modulation by FBP and ATP indicate that it is a normal, positively co-operative enzyme. The apparent negative co-operativity of the
female enzyme might be an adaptation to maintain a more steady rate
of glycolysis. Conway & Koshland (1968) have suggested that negative
co-operativity tends to insulate an enzyme from fluctuations in
metabolite concentration. These points are supported by the ATP
and AMP levels in these parasites, the significance of which is
discussed in the metabolite section (4.3). Modulation of the
parasite enzymes occurred within the physiological concentration
ranges determined from the metabolite levels.
With regard to the other species investigated, all the PK saturation curves were sigmoidal and positively co-operative except
S.bovis, for which the data was incomplete. It should be borne in
mind however, that co-operativity does not necessarily indicate regulatory capabilities and the high Km values of the enzymes cast
doubt on their regulatory capacities. Nevertheless, it would seem
that the strain of female S.mansoni used in the present study is
exceptional in its regulation of glycolysis. The apparent KrmKT)
for the enzyme is higher than that found by Brazier & Jaffe (1973)
157
while the male K ’ is lower,cthus stressing the difference in proper
ties between the enzymes.A number of points should be borne in mind when transposing
in vitro data to an in vivo situation. Firstly, the present study
deals only with crude homogenates and the assay system may have been
subject to extraneous enzyme interference but this must be similar
to the system which operates in vivo. Analysis of enzyme activity
in crude homogenates also makes no allowance for possible internal
compartmentalisation of enzymes. It may be that isoenzymes with
different kinetic properties have a differential specific distri
bution throughout the parasite and normal analysis would only yield an average of these activities. Secondly, homogenates will contain
small quantities of endogenous metabolites which will alter assay concentrations but the available data indicates that the maximum
error will only be approximately 2%. Additionally, the measurement of enzyme activity at saturating conditions of reactants will cause
the enzyme to behave in an ’’unphysiological” manner thus creating
the possibility of drawing incorrect conclusions regarding the manner
of its ijn vivo function.
Determination of enzyme.activity at ’’physiological” levels of
metabolites is at least a closer approximation of the in vivo func
tioning of the enzyme but requires a preliminary study of the metabolites involved. There are still a number of problems that must be
considered when performing this type of analysis. The measurement of the intracellular concentration of participating metabolites can
only be an approximation due to the inability to remove all the
extracellular fluid that is present. Also, the schistosome gut
contains host (erythrocyte) enzymes which may be active. However,
the present study indicates that interference from this source is minimal (Section 3.11.5.1.).
Another important factor to consider is that sigmoidal curve does not automatically imply a co-operative or regulatory nature.
A two substrate enzyme mechanism could also account for a sigmoidal
shape (Price & Stevens, 1982). In S.marqrebowiei, male and female
PKs showed co-operative kinetics and were sensitive to modulation
by FBP and ATP. The effective range of ATP inhibition for the male
enzyme was between 1nM - 1jjM which is well below the range for male
S.mansoni PK. The steady state levels of ATP were not measured in
S.marorebowiei but as both sexes produced more lactate during incubation than S.mansoni and showed similar glycolytic enzyme activities, it seems reasonable to suggest that ATP levels in the
parasites would be higher than Ijjffl. The female enzyme was inhibited
within the 1pM - ImM ATP range and thus shows better agreement with the estimated endogenous levels.
Male 5.marqrebowiei PK was more significantly activated by
FBP than the female enzyme from 1nM - 1(jM but the endogenous levels
in males and females were 14 jjM and 28 jjM respectively. In this range
(1 jjM - ImM) the female enzyme was more significantly activated than
the male. The male worms do not appear to require a greater rate of
glycolysis than^.the females. This may be because they are less motile
in vivo than male S.mansoni or they may have other means of generating
ATP such as the utilisation of a more active TCA cyiJe..Measurements of the "physiological” behaviour of S.mansoni PK
showed that FBP activation occurred in both sexes but activation of
the male enzyme was significantly higher than the PK from the females
by a factor of 8. Female PK was not significantly inhibited by ATP
in the absence of FBP and this finding is similar to the result
obtained from the assays performed under saturating metabolite con
ditions. However, in the presence of FBP, which is more representative of the in vivo situation, female PK activity was inhibited to the same extent as the male enzyme. This highlights the potential dangers
of drawing incorrect conclusions from results obtained under non-
physiological conditions. As no sigmoidal relationship was established
for either enzyme it was impossible to determine the extent of co-
operativity.The greater effect of FBP on male PK may be seen as a reflection
of the probable in vitro mode of life of the adult parasites. Male
worms normally carry the female parasites along the hosts’ mesenteric/ portal/urinary blood vessels, sometimes against the flow of blood.
Therefore, it seems reasonable that the males would be required to
produce periodic bursts of energy which could be provided by a large increase in glycolytic rate which in turn could be mediated via FBP.In addition, the greater rate of glycolysis in the males could well be the reason why they contain greater glycogen levels than females.In contrast, the adult females have to perform comparatively little mechA
nical activity and so would not need to alter their glycolytic rate and
170
s
*
m
&
♦
The data regarding ATP and AMP levels in S.mansoni also indicates
that the females have a lomer, steadier rate of glycolysis than the males. The higher ATP levels and ATP : AMP ratio in the male parasites indicate that the rate of energy utilisation and rate of change
of the glycolytic flux is higher than in the females (Beis & Nemsholme
1975). This information supports the idea that male morms mould
require periodic bursts of energy production to hold the females
mithin the gynecophoric canal and to transport them about the hosts1
blood system.
The presence of FBP in assay measurements is a more accurate
representation of the in vivo situation and under such conditions ATP
inhibited male PK activity uncompetitively and female PK competitively.
This suggests a gender based difference in glycolytic control but
as the data did not conform precisely to the normal Linemeaver/Burk transformations, further mork is necessary to confirm this finding.
Male and female PK shomed no preference for Mg or Mn mhereas
Brazier & Jaffe (1973) found that Mn"mas half as affective as Mg’
in terms of enzyme activity. The Mg concentration used in the assay
(5mM) mas reasonably close to the physiological level (approximately
3mM) reported by Sham & Erasmus (1983). In contrast, the PKs from other parasites such as the daughter sporocysts of Microphallus
similis (McManus & James, 1975), Dirofilaria immitis (Brazier &
Jaffe, 1973) and Moniezia expansa (Bryant, 1972) do exhibit a pre-~H* 4fference for Mg or Mn. Yet these preferences may be related to the
so mould require lomer glycogen stores than the males.
in vitro assay conditions as little is knomn concerning the inorganic
1 I I
metabolism of these parasites. The preferred cation may not even be
available in vivo to the parasite enzyme. These observations high
light the difference between the enzyme in the present study and
the enzyme examined by Brazier & Jaffe (1973). Apart from strain
variation, differences in buffer systems and pHs could also account,
in part, for this variation.
Electrophorectic analysis revealed that PK exists in identical
fprms in male and female worms although the number of separated
bands, which may represent discrete isoenzymes, varied according, to the technique employed. Each method of analysis indicates that there is one major form of the enzyme in both sexes with a number of
minor isoenzymes. Mammalian tissues contain at least two distinct
PK isoenzymes which possess different kinetic characteristics (Tanaka
et al 1965, 1967). Type L PK is activated by FBP, is sensitive to
ATP inhibition (Tanaka et al, 1967) (li/eber, 1967) and is predominant
in the liver. Type M PK is found in skeletal muscle, is not activated
by FBP and is far less sensitive to ATP inhibition (Tanaka et_ al,
1967) (Vijayvarqiya et al 1969). Brazier & Jaffe (1973) found that
paired S.mansoni PK resembled Type M PK but the enzymes in the present
study resemble Type L. It is not known if the isoenzymes (sub-units) found in the present study have different kinetic properties but the
isoelectric point measurements will serve as a baseline for the isolation and purification of the enzyme in any subsequent study.
Regulation of glycolysis in S.mansoni is thought to be mediated via hexokinase (HK) (Bueding & Makinnon, 1955a) (Shapiro & Talalay,
1982b), PFK (Bueding & Fisher, 1966) and possibly glucosephosphate
172
#
isomerase (GPI) (Shapiro & Talalay, 1982a). HK and PFK are the
classical regulators of glycolysis, typically catalysing non- equilibrium reactions in vivo and have relatively low specific activities (Price & Stevens, 1982). Traditionally the role of PK
in regulation of glycolysis has been doubtful due to its relatively
high specific activity (Price & Stevens, 1982). Nevertheless, in the
present study the mass action ratios and kinetic study has shown that
PK can control glycolytic flux. HK and PFK ultimately control the
rate of entry of glucose along the pathway but PK would regulate the
rate of lactate formation, assuming that lactate dehydrogenase is not
allosterically controlled. This would be important in terms of energy
synthesis and will be discussed below. Also, the control of PK may
be essential to maintain sub-toxic levels of lactate in the tissues.
PK could also help to maintain cytoplasmic redox balance and it may also allow a small proportion of phosphoenolpyruvate (PEP) and pyruvate to be converted to amino acids (Conn & Stumpf, 1976) or
the former to participate in CD^ fixation reactions.
PK is an extremely important enzyme in glycolysis as it determines the net ATP synthesis by the pathway. Fructose-6-phosphate
(F-6-P) requires ATP for the phosphorylation step to FBP and assuming
that endogenous glucose also requires ATP to be phosphorylated by
HK, then the synthesis of ATP from 1, 3-diphosphoglycerate cle^age will result in an overall balance. To gain energy (ATP) from the
system, the clevage of phosphate from PEP is necessary and for a parasite so heavily dependant on glycolysis, regulation of the net gain step in the system via PK would seem to be essential. A further gain
173
of ATP can be achieved by catabolising glucose-1-phosphate derived
from glycogen breakdown but the relative rates of endogenous and
exogenous glucose utilisation are not known.In summary, the PKs from the strain of S.mansoni used for the
present study, appear to be capable of regulating glycolysis.Control of enzyme activity in both sexes is mediated via the same
modulators although the male enzyme shows an enhanced response,
which is probably correlated to its in vivo life style. A comparative
kinetic study of purified PKs from male and female worms may afford a greater insight as to the mechanism of regulation, providing
"physiological” levels of metabolites are employed. Interspecific
comparisons may also be valuable as differences in control mechanisms
may offer the possibility of exploitation by chemotherapy.
4.6. OXYGEN UPTAKE AND CYTOCHROMES
The physiological role of oxygen in schistosome metabolism has
been the subject of debate for a number of years. Schiller et al
(1975) found that oxygen was necessary for egg production, presumably
for the tanning of egg shells (Seed et al, 1978). Bueding & Charms (1952) failed to find sufficient quantities of cytochrome C and
cytochromecC oxidase in S.mansoni to account for more than one tenth of the parasites’ oxygen uptake. Bueding (1950) found that male and
female worms took up quantities of oxygen which could account for
only 3% of the total glucose utilisation. Hence, this evidence suggests that oxidative phosphorylation is operating negligibly, if
at all in S.mansani.
In contrast, Coles (1972b) reported that oxidative phosphory
lation provides at least one quarter of the worms’ metabolic energy. He also suggested that this was an underestimate of the in vivo
capacity. It is unfortunate that this data refers only to paired
schistosomes as the study of separate oxygen uptake rates for male
and female worms’ would facilitate a better estimate of the contri
bution made by egg production to oxygen uptake.
The present study revealed that female S.mansoni took up eight
times as much oxygen as the male parasites. The values were higher
than those obtained by Bueding (1950) and Bueding & Charms (1952)
and this is probably due to the more physiological medium and more
sensitive polarographic techniques used in this study. 20 mM KCN completely inhibited oxygen uptake by the males but only inhibited
the females’ uptake by 64%. Ross & Bueding (1950) found that com
plete inhibition of uptake in both sexes occurred in the presence
of ImM KCN. Therefore, assuming that KCN is only uncoupling
cytochrome oxidase, then it appears that both sexes can utilise
oxidative phosphorylation for energy production. The presence of
KCN greatly reduced the worms’ motility but did not kill them.
Coles (1972b) reported a concomitant increase in lactate production
in the presence of cytochrome chain uncouplers. The females’
continuing ability to take up oxygen in the presence of KCN could be consistent with a requirement for egg shell production. Another possibility is that the female parasites possess an alternative electron transport chain that is cyanide insensitive. The data
175
w*dL\Cccfe,$ that ATP synthesis by oxidative phosphorylation important to the female parasites than to the males. Morris &
Threadgold (1968) reported that in both sexes the majority of the
mitochondria are found in the uiorms1 parenchyma. Numerous mitochondria are found surrounding oocytes (Roberts, 1983), therefore
oxidative phosphorylation in the female worms may be used to provide
energy for egg synthesis as well as for general metabolism.
Until the present study it has been generally accepted that
measurement of the cytochrome spectra of adult schistosomes was not
possible due to interference by the parasites' gut contents (Coles,
1982). Cheah (1975) used nitrate in an attempt to make a clearer
distinction between Ascaris cytochrome spectra and absorbance due
to haemoglobin. The present study revealed that haematin (a haemoglobin degradation product) and not whole mouse blood is the major
contaminant in cytochrome scans. Also, by performing assays at
-196°C there is an intensification factor of 7 for cytochromes only
and hence is more sensitive than room temperature assays (Wilson, 1967). The present investigation has shown that the cytochrome
spectra of schistosomes can be measured.
Comparisons of parasite spectra with those from rat liver shows
that male and female S.mansoni have identical spectra corresponding
to cytochromes a^, C, and C^. Male parasites also possessed a 'b* type cytochrome which was not detectable in the females. This may
have been due to insufficient material being used in the assays.
However, the absence of cytochrome b in female schistosomes, together
with the degree of KCN insensitivity that was found by oxygen uptake
measurements may indicate a branched respiratory chain. Other para
sites eg A.lumbricoides, Moniezia expansa, Haemonchus contortus and Fasciola hepatica are thought to operate branched cytochrome chains,
with an 0 type cytochrome acting as a terminal oxidase (Barrett,
1981).
The quantities of cytochromes present in both sexes of S.mansoni
are similar therefore if the females are more dependent on oxidative
phosphorylation then some form of restriction or control must occur
in the males. Bryant (1970) found that an oxygen tension of 5 mm Hg
was necessary for normal electron transport and the oxygen tension
of venous blood (49 - 66 mm Hg) is well above this value (Smyth, 1976). Oxygen would presumably be available to the parasites from digested
haemoglobin. Hence, as the male digestive tract contains smaller quantities of digested material than the female, it is possible that
limitation of quantity of oxygen available is a reason for the males'
apparent lower rate of electron transport. However, in vitro, it may
be possible that transtegumental absorption of oxygen occurs in which
case another means of regulation may apply..
Control of electron flux into the mitochondria could be anothermechanism for regulating oxidative phosphorylation. It is not knownhow the transport chains in these parasites are linked to redox balance.
Coupling of the chain may be effected by a malate-asparate shuttle,glycerol
a glycerol shuttle or a TCA cycle. The ' shuttle system, would result in a slightly reduced ATP output as tMs would presumably link at the ubiquinone level and so lose a site 1 phosphorylation.
The feasibility of the existence of these systems in schistosomes
is discussed in the incubation section (4.4).
Electron chain coupling via a TCA cycle must remain a doubtful prospect as the existence of a functional cycle has yet to be clearly
demonstrated in the parasite strains used in the present study.
S.marqrebowiei appears to be capable of operating a cycle (see section
4.2) and even if S.mansoni has a functional pathway, the estimated rates of cycle turnover in these species indicates that it would not
make a significant contribution to ATP synthesis in these worms.
Clearly, the mechanism of cytochrome coupling to redox reactions in
these parasites warrants further investigation.
Huang (1980) found that S.japonicum relied substantially on
oxidative phosphorylation as a means of energy synthesis. He also
found that oxygen consumption was markedly affected by the composi
tion of the incubation medium used for the experiments. The presence
of serum was found to be essential to maintain the worms in an active state for longer than 2 - 3 hours. Rogers (1976) found that
S.douthitti was capable of KCN sensitive and insensitive aerobic metabolism and so, electron transport mechanisms may be more wide
spread and varied in schistosomes than has previously been suggested. The quantities of cytochromes found in 5,mansoni are similar to those
found in Ascaris and Moniezia (Cheah, 1972) and interestingly, are
higher than those found in highly aerobic sea urchin sperm (Wilson &
Epel 1978). Cercarial cytochromes were measured by Coles (1972a)
and Coles & Hill (1972) who detected cytochromes a/a^, b, C and
possibly 0 but quantitative analyses were not performed. The spectral
peaks are different to those obtained in the present study because
the assays were performed at room temperature.If a more active TCA cycle was operating in S.mansoni, then as
6 moles of oxygen are required for the complete oxidation of 1 mole
of glucose, the oxygen uptake rates would account for the oxidation
of 0*1 and 0*7 moles of glucose for males and females respectively. These values would account for the "excess" glucose consumption shown particularly by the female worms. However, all the data indicates that
TCA operation in these parasites is at best, minimal. Nevertheless,
this does not necessarily mean that glucose consumption is not linked to the electron transport chain. A malate or glycerol shuttle opera
ting at a high turnover could be equally as efficient in supplying
electrons for oxidative phosphorylation. As glyceraldehyde-3-phosphate
dehydrogenase (GPBB) activity in S.mansoni was far higher than PEPCK
activity, a glycerol shuttle mechanism would seem more appropriate for
chain coupling than a malate shuttle supplied via CO^ fixation. This
would mean that part of the glucose flux would be diverted from lactate production into synthesis of dihydroxyacetone phosphate (DHAP) and
glycerol-3-phosphate (G-3-P). If a large proportion of the "excess"
glucose is being metabolised in this manner then it would result in relatively high levels of these intermediates. This is supported by the fact that DHAP levels were relatively higher than levels of other
glycolytic intermediates in female S.mansoni. A similar situation did not exist for DHAP levels in male S.mansoni and hence this fits the overall picture of the females being more dependant on oxidative
phosphorylation than the males.
DHAP levels alone in female S.mansoni can account for 20% of the
"excess" glucose (0«6 pmoles) consumed by these worms. Determination
of glycerol-3-phosphate levels in the females may yield similar
values. Therefore the levels of intermediates necessary to operate
a glycerol shuttle could account for nearly half the extra glucose
consumption. Hence a significant proportion of the glucose consump
tion by female S.mansoni not channelled into lactate production could
be accounted for by oxidative phosphorylation coupled via a shuttle
mechanism.Further work is necessary to substantiate this idea but it is
evident from the levels of cytochromes and oxygen uptake rates of
these parasites, that electron transport chains^play an important role in energy production in S.mansoni whatever the means of redox
coupling is. Other species of schistosome with greater levels of
"excess" consumed glucose eg S.haematobium could well be even more
dependent on oxidative phosphorylation than S.mansoni. An inter
specific comparison of this aspect of metabolism may yield interesting
results. The pathway suggested for S.mansoni females does not appear
to fit the females of S.marqrebowiei as DHAP levels were not detectable
in the latter. This species however, appears to be capable of TCA
cycle activity and so a different means of chain coupling could exist
in this case which could even augment other mechanisms of redox
coupling.
If schistosomes are capable of aerobic metabolism then a Pasteur effect should be demonstrable. Bueding (1950), Bueding & Fisher (1966)
and Schiller et al (1975) could find no evidence of this but such
corroborating data was found by Coles (1972b). Again, the choice
of medium or parasite species/strain may lead to different results
but it is obvious that the potential for aerobic metabolism by some, if not all schistosomes, cannot be ignored.
4.7. SUMMARY
Studies of the glucose, glycogen, protein and wet weight levels in
both sexes of S.mansoni and S.marqrebowiei indicated that with the
exception of male S.marqrebowiei, the parasites had levelled off
their growth rates by 56 - 70 days post infection. Male S.marqrebowiei continued to grow and this may be due to the increasing synthesis and
storage of glycogen within the worm.Incubation studies, metabolite and enzyme analyses indicate
that glycolysis is a major pathway of ATP synthesis in schistosomes.The evidence for the functioning of a TCA cycle in S.mansoni is
inconclusive but the data suggests that S.marqrebowiei does operate a cy cle. However, the studies showed that the TCA cycle contributes little,
if at all, to ATP production. HMP shunt enzymes were present in both
species but again, there is no.data to suggest that the pathway is fully functional in these parasites.
The incubation work revealed that whilst male S.mansoni and S.marqrebowiei are possibly homolactic fermenters, the females channel approximately half their glucose consumption into other
metabolic pathways. Part of the excess glucose may be channelled into egg production, tegument formation, synthesis of circulating
antigen or possibly neutral lipid synthesis. Alternatively a; pro
portion of the glucose may be linked to oxidative phosphorylation mechanisms.
Oxygen uptake measurements and cytochrome spectra analyses
indicate that both sexes of S.mansoni are^capable of oxidative phos=« phorylation. The females, however, appear to be more reliant on this
process than the males. Metabolite and specific enzyme analysis suggest that the cytochrome chain is coupled to the redox balance via
a glycerol shuttle mechanism. Additionally,.female S.mansoni also
showed a degree of cyanide insensitivity which could mean that the
oxygen is possibly being used for egg shell tanning or that a branched
cytochrome chain is present.
Enzyme kinetic studies, supported by metabolite and electrophoretic analyses, indicated that the pyruvate kinases from male and
female S.mansoni are structurally similar and exert a regulatory influence on the parasites’ glycolytic flux in vivo. The enzyme
from the males was 8 times more sensitive to FBP activation than the female enzyme. This may be necessary to allow the males to meet the
changes in energy demand placed on them by their in vivo life style.
The PKs of both sexes was inhibited by ATP to the same degree.
Studies on the PKs from S.margrebowiei and 5.japonlcum revealed that
the enzymes are positively co-operative but it is not certain if they are capable of regulating glycolysis in these species.
In general, the schistosomes studied, particularly the males, appeared to be heavily dependent on anaerobic glycolysis for energy
132
production. However, the females of certain species (and both sexesof 5.haematobium) appear capable of other means of ATP synthesis. It
is obvious that female S.mansoni have a large potential for aerobic
energy production. The same remains to be demonstrated in otherAspecies. This may prove to be an important co^ideration when exam
ining new approaches to the chemotherapy of schistosomiasis.
♦
*►
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